Hepatitis C virus immunogenic compositions and methods of use thereof

ABSTRACT

The present disclosure provides immunogenic compositions comprising: a) hepatitis C virus (HCV) E1E2 heterodimers, HCV E2, or HCV E1; and b) an adjuvant, where the adjuvant is a cyclic dinucleotide or an archaeosome. The present disclosure provides methods of inducing an immune response in an individual to HCV, the methods comprising administering to an individual an effective amount of an immunogenic composition of the present disclosure.

CROSS-REFERENCE

This application is a national stage filing under 35 U.S.C. § 371 ofPCT/CA2017/051192, filed Oct. 5, 2017, which claims the benefit of U.S.Provisional Patent Application No. 62/406,770, filed Oct. 11, 2016,which applications are incorporated herein by reference in theirentirety.

INTRODUCTION

Hepatitis C virus (HCV) is a blood-borne pathogen that is estimated toinfect 150-200 million people worldwide. Infection by HCV may benon-symptomatic, and can be cleared by patients, sometimes withoutmedical intervention. However, the majority of patients develop achronic HCV infection, which may lead to liver inflammation, scarring,and even to liver failure or liver cancer. In the United States alone,over 3 million people have a chronic infection.

The HCV virion contains a positive-sense single stranded RNA genome ofabout 9.5 kb. The genome encodes a single polyprotein of 3,010 to 3,030amino acids. The structural proteins comprise a core protein forming theviral nucleocapsid and two envelope glycoproteins, E1 and E2.

A vaccine based on the recombinant envelope glycoproteins (rE1E2) from asingle genotype 1a strain (HCV-1) protected chimpanzees from chronicinfection following homologous and heterologous genotype 1a (gt1a) viralchallenge (reviewed in Houghton, M Immunol Rev 2011). Antisera from theimmunized chimpanzees were shown to exhibit in vitro cross-neutralizingactivity (Meunier et al. (2011) J. Infect. Dis. 204:1186). A phase Iclinical trial was conducted in human volunteers with a similar antigen(Frey et al. (2010) Vaccine 28:6367). Antisera from selected vaccinatedindividuals were similarly capable of neutralizing chimeric cellculture-derived viruses (HCVcc) expressing the structural proteins ofstrains representing all 7 major HCV genotypes in vitro (Law et al.(2013) PLoS One 8:e59776) and to be able to compete with the binding ofnumerous discrete monoclonal antibodies with broad cross-neutralizingactivities (Wong et al. (2014) J. Virol. 88:14278).

There is a need in the art for compositions and methods for inducingimmune responses to HCV.

SUMMARY

The present disclosure provides immunogenic compositions comprising: a)hepatitis C virus (HCV) E1E2 heterodimers, HCV E2, or HCV E1; and b) anadjuvant, where the adjuvant is a cyclic dinucleotide or an archaeosome.The present disclosure provides methods of inducing an immune responsein an individual to HCV, the methods comprising administering to anindividual an effective amount of an immunogenic composition of thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C provide an amino acid sequence alignment of examples of thecore-E1-E2 coding regions of a HCV genotype 1 virus, specificallyrepresentative HCV 1A, 1B and 1C genotypes. Genbank database sequencesfor the coding region core-E1-E2 were aligned using Geneious softwarev5.6.4. Numbering of amino acids is according to strain NP_671941 (H77).Consensus: SEQ ID NO:1; AVI1a129: SEQ ID NO:2; NP_671491 (H77): SEQ IDNO:3; EU155269: SEQ ID NO:4; EU781810: SEQ ID NO:5; EU781771: SEQ IDNO:6; AB250610: SEQ ID NO:7; EU781752: SEQ ID NO:8; EU781759: SEQ IDNO:9; EF407439: SEQ ID NO:10; EF407427: SEQ ID NO:11; EU362905: SEQ IDNO:12; EF407413: SEQ ID NO:13; EU781808: SEQ ID NO:14; EU78170: SEQ IDNO:15; AJ238799 (Con1): SEQ ID NO:16; AAK97744: SEQ ID NO:17; AF139594:SEQ ID NO:18; AF176573: SEQ ID NO:19; BAA19625: SEQ ID NO:20; BAA25076:SEQ ID NO:21; BAC54896: SEQ ID NO:22; BAD91386: SEQ ID NO:23; BAF46764:SEQ ID NO:24; BAG30950: SEQ ID NO:25; CAB41951: SEQ ID NO:26; AAK95832:SEQ ID NO:27; AAT69968: SEQ ID NO:28; and BAA03581: SEQ ID NO:29.

FIG. 2A-2C provide an alignment of amino acid sequences of thecore-E1-E2 coding region of representative HCV 2A and HCV2B subtypes.Genbank database sequences for the coding region core-E1-E2 were alignedusing Geneious software v5.6.4. The amino acid numbering depicted is inaccordance to the common HCV strains: AB047639 (JFH1) and HPCJ8G-J8 (J8)for HCV2A and HCV2B, respectively. AB047639 (JFH1): SEQ ID NO:30;AB047645: SEQ ID NO:31; AF169003: SEQ ID NO:32; AF169005: SEQ ID NO:33;AF238482: SEQ ID NO:34; AY746460: SEQ ID NO:35; HPCPOLP: SEQ ID NO:36;NC_009823: SEQ ID NO:37; HPCJ8G HC-J8: SEQ ID NO:38; AB030907: SEQ IDNO:39; AY232730: SEQ ID NO:40; AY232747: SEQ ID NO:41; and DQ430817: SEQID NO:42.

FIG. 3A-3C provide an amino acid sequence alignment of the core-E1-E2coding region for representative HCV 3A, 3B and 3K genotypes. Genbankdatabase sequences for the coding region core-E1-E2 were aligned usingGeneious software v5.6.4. Consensus: SEQ ID NO:43; AVI3a177: SEQ IDNO:44; YP_0014696: SEQ ID NO:45; CAA54244: SEQ ID NO:46; AAC03058: SEQID NO:47; AAY29642: SEQ ID NO:48; ABD85062: SEQ ID NO:49; ABD85063: SEQID NO:50; ABD97104: SEQ ID NO:51; BAA06044: SEQ ID NO:52; BAA08372: SEQID NO:53; and BAA09890: SEQ ID NO:54.

FIG. 4A-4B provide an amino acid sequence of the core-E1-E2 codingregion for HCV genotype 7a. Amino acid sequence for the coding regioncore-E1-E2 of genotype 7a (isolate QC69; Genbank: ABN05226.1; SEQ IDNO:55) is shown according to the numbering scheme of the referencestrain, NP_671941 (H77).

FIG. 5A-5C provide amino acid sequences of immunoglobulin Fc regions forGenBank 3S7G_A Homo sapiens IgG1 Fc: SEQ ID NO:56; GenBank AAN76044 Homosapiens IgG2 Fc: SEQ ID NO: 57; GenBank AAW65947 Homo sapiens IgG3 Fc:SEQ ID NO:58; GenBank AAA52770 Homo sapiens IgD Fc: SEQ ID NO:59;GenBank 0308221A Homo sapiens IgM Fc: SEQ ID NO:60; GenBank P01876 Homosapiens IgA Fc: SEQ ID NO:61; GenBank IF6A_B Homo sapiens IgE Fc: SEQ IDNO:62; and GenBank P01861 Homo sapiens IgG4 Fc: SEQ ID NO:63.

FIG. 6 presents Table 1, which provides conserved regions based onconserved CD4 epitopes (CD4⁺ T cell epitopes) conserved among HCVgenotypes. Top to Bottom: SEQ ID NOs.:64-74.

FIG. 7 presents Table 2, which provides the number of located HCV CD8⁺ Tcell epitopes and anchor positions for common human leukocyte antigen(HLA)-I Alleles in the United States.

FIG. 8 presents Table 3, which provides conserved regions based on CD8epitopes (CD8⁺ T cell epitopes) conserved among HCV genotypes. Top toBottom: SEQ ID NOs.:75-84.

FIG. 9A-9B provide a list of CD4 and CD8 epitopes that are conservedamong HCV genotypes 1a, 1b, 2a, 2b, and 3.

FIG. 10A-10D provide amino acid sequences of examples of polypeptidescomprising multiple T-cell epitopes (TP29: SEQ ID NO:85; TP50: SEQ IDNO:86; TP52: SEQ ID NO:87; TP70: SEQ ID NO:88; TP100: SEQ ID NO:89;TP171: SEQ ID NO:90; TP228: SEQ ID NO:91; TP553: SEQ ID NO:92; TP778:SEQ ID NO:93; and TP1985: SEQ ID NO:94). The start and end amino acidsare based on the sequence designated “Consensus” in FIG. 12A-12L. TheT-cell epitopes contained within each polypeptide are provided; theT-cell epitope designations correspond to those presented in FIG.11A-11N.

FIG. 11A-11N provide consensus amino acid sequences of HCV polypeptides;and depict the locations of T-cell epitopes (SEQ ID NO:95).

FIG. 12A-12L provide consensus amino acid sequences of HCV polypeptides(SEQ ID NOs.96-107).

FIG. 13 depicts the experimental protocol.

FIG. 14 depicts neutralization activity of recombinant E1E2 (rE1E2), incombination with adjuvant, in mice.

FIG. 15 depicts differential detection of T cell immune responses inmice.

DEFINITIONS

The term “hepatitis C virus” (“HCV”), as used herein, refers to any oneof a number of different genotypes and isolates of hepatitis C virus.Thus, “HCV” encompasses any of a number of genotypes, subtypes, orquasispecies, of HCV, including, e.g., genotype 1, 2, 3, 4, 6, 7, etc.and subtypes (e.g., 1a, 1b, 2a, 2b, 3a, 4a, 4c, etc.), and quasispecies.Representative HCV genotypes and isolates include: the “Chiron” isolateHCV-1, H77, J6, Con1, isolate 1, BK, EC1, EC10, HC-J2, HC-J5; HC-J6,HC-J7, HC-J8, HC-JT, HCT18, HCT27, HCV-476, HCV-KF, “Hunan”, “Japanese”,“Taiwan”, TH, type 1, type 1a, H77 type 1b, type 1c, type 1d, type 1e,type 1f, type 10, type 2, type 2a, type 2b, type 2c, type 2d, type 2f,type 3, type 3a, type 3b, type 3g, type 4, type 4a, type 4c, type 4d,type 4f, type 4h, type 4k, type 5, type 5a, type 6 and type 6a.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, non-human primates (e.g., simians), equines (e.g., horses),rodents (e.g., rats; mice), and humans.

As used herein, the term “isolated,” in reference to a polypeptide,refers to a polypeptide that is in an environment different from that inwhich the polypeptide naturally occurs. An isolated polypeptide can bepurified. By “purified” is meant a compound of interest (e.g., apolypeptide) has been separated from components that accompany it innature. “Purified” can also be used to refer to a polypeptide separatedfrom components that can accompany it during production of thepolypeptide (e.g., during synthesis in vitro, etc.). In someembodiments, a polypeptide (or a mixture of polypeptides) issubstantially pure when the polypeptide (or mixture of polypeptides) isat least 60% or at least 75% by weight free from organic molecules withwhich it is naturally associated or with which it is associated duringproduction. In some embodiments, the polypeptide is from 30% to 60%pure. In some embodiments, the polypeptide (or mixture of polypeptides)is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, or at least 99%, by weight, pure. For example, in someembodiments, an E1 or an E2 polypeptide (or a mixture of E1 and E2polypeptides, e.g., an E1/E2 heterodimer) is substantially pure when theE1 or E2 polypeptide (or mixture of E1 and E2 polypeptides) is at least60% or at least 75% by weight free from organic molecules with which thepolypeptide(s) is naturally associated or with which it is associatedduring production. In some embodiments, the E1 or E2 polypeptide (ormixture of E1 and E2 polypeptides) is at least 60%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%, byweight, pure. In some embodiments, where a composition comprises an E2polypeptide, the E2 polypeptide is at least 60%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99%, byweight, pure. In some embodiments, where a composition comprises anE1/E2 heterodimeric complex polypeptide, the E1/E2 heterodimeric complexpolypeptide is at least 60%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 99%, by weight, pure. In someembodiments, where a composition comprises a T-cell epitope polypeptide,the T-cell epitope polypeptide is at least 60%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99%, byweight, pure.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases. In some cases, apolynucleotide is RNA. In some cases, a polynucleotide is DNA. A“polynucleotide” includes a nucleic acid that is incorporated into aviral vector or a bacterial vector.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term “polypeptide”includes glycosylated polypeptides.

The term “heterologous” refers to two components that are defined bystructures derived from different sources. For example, where“heterologous” is used in the context of a polypeptide, where thepolypeptide includes operably linked amino acid sequences that can bederived from one or more different polypeptides, e.g., amino acidsequences that are not operably linked to the polypeptide in nature. Asanother example, where a composition comprises an HCV E1/E2 heterodimerand a “heterologous” polypeptide, the “heterologous polypeptide is apolypeptide other than HCV E1 or HCV E2. As another example, where acomposition comprises an HCV E1 polypeptide and a “heterologous”polypeptide, the “heterologous polypeptide is a polypeptide other thanHCV E1. As another example, where a composition comprises an HCV E2polypeptide and a “heterologous” polypeptide, the “heterologouspolypeptide is a polypeptide other than HCV E2.

The term “archaeal lipid” refer to a polar lipid common to the DomainArchaea typified by isoprenoid chains in ether linkage to the sn-2,3carbons of the glycerol backbone.

Archaeal core lipids are most commonly 2,3-di-O-sn-diphytanylglycerol(archaeol), and 2,2′,3,3′-tetra-O-dibiphytanyl-sn-diglycerol(caldarchaeol).

Synthetic archaeal lipids or polar synthetic lipids refer to core lipidprecursors either derived from Archaeal lipids by hydrolysis or made bychemical synthesis, conjugated to at least one new head group.

Archaeol phospholipids are referred herein to using archaetidyl, forexample, AG, archaetidylglycerol; AS, archaetidylserine.

The term “conventional lipids” refers to the lipids common to theDomains Bacteria and Eukarya. This includes polar lipids typified byfatty acyl chains in ester linkage to the sn-1,2 carbons of the glycerolbackbone, and neutral lipids such as cholesterol. Conventionalphospholipids are referred to in the usual way, for example, DPPG,dipalmitoylphosphatidylglycerol; DPPS, dipalmitoylphosphatidylserine.

The term “archaeosome” refers to a closed lipid vesicle that containsany amount of synthetic archaeal lipid(s).

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anHCV E1E2 heterodimer” includes a plurality of such heterodimer andreference to “the cyclic dinucleotide” includes reference to one or morecyclic dinucleotides and equivalents thereof known to those skilled inthe art, and so forth. It is further noted that the claims may bedrafted to exclude any optional element. As such, this statement isintended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides immunogenic compositions comprising: a)hepatitis C virus (HCV) E1E2 heterodimers, HCV E2, or HCV E1; and b) anadjuvant, where the adjuvant is a cyclic dinucleotide or an archaeosome.The present disclosure provides methods of inducing an immune responsein an individual to HCV, the methods comprising administering to anindividual an effective amount of an immunogenic composition of thepresent disclosure.

Immunogenic Compositions Comprising HCV E1E2, E2, or E1Polypeptide+Cyclic Dinucleotide

The present disclosure provides an immunogenic composition comprising:a) an HCV E1E2 heterodimer; and b) a cyclic dinucleotide (CDN). Thepresent disclosure provides an immunogenic composition comprising: a) anHCV E2 polypeptide; and b) a CDN. The present disclosure provides animmunogenic composition comprising: a) an HCV E1 polypeptide; and b) aCDN.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces an immuneresponse in the individual to one or more HCV genotypes. In some cases,an immunogenic composition of the present disclosure, when administeredto an individual in need thereof, induces an immune response in theindividual to one or more HCV genotypes, where the immune response isgreater than the immune response induced by administration of a controlcomposition comprising the HCV E1/E2 heterodimer (or E1 polypeptide, orE2 polypeptide) but lacking the CDN.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces CD8⁺ CTLsspecific for HCV, where the number of HCV-specific CD8⁺ CTLs induced isat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, higher than the number of HCV-specific CD8⁺ CTLs induced byadministration of a control composition (e.g., a composition comprisingthe HCV E1/E2 heterodimer but lacking the CDN; a composition comprisingan E1 polypeptide but lacking the CDN; a composition comprising an E2polypeptide but lacking the CDN).

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces CD4⁺ T cellsspecific for HCV, where the number of HCV-specific CD4⁺ T cells inducedis at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, higher than the number of HCV-specific CD4⁺ T cells induced byadministration of a control composition (e.g., a composition comprisingthe HCV E1/E2 heterodimer but lacking the CDN; a composition comprisingan E1 polypeptide but lacking the CDN; a composition comprising an E2polypeptide but lacking the CDN).

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces productionof HCV-specific CD4⁺ T cells and CD8⁺ T cells in the individual, wherethe number of HCV-specific CD4⁺ T cells and/or CD8⁺ T cells isincreased, such that the percent of total peripheral CD4⁺ and/or CD8⁺ Tcells that is HCV-specific is from 0.01% to 0.05%, from 0.05% to 0.10%,from 0.10% to 0.125%, from 0.125% to 0.25%, from 0.25% to from 0.50%, or0.5% to 10% (e.g., from 0.5% to 1%, from 1% to 2%, from 2% to 5%, orfrom 5% to 10%). The number of HCV-specific CD4⁺ T cells and CD8⁺ Tcells in a control individual (e.g., an individual not infected withHCV) not treated with the immunogenic composition would be undetectable.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, increases the numberof HCV E1/E2-specific CD4⁺ T cells and CD8⁺ T cells in the individual byat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, compared to the number of HCV E1/E2-specific CD4⁺ T cells andCD8⁺ T cells in the individual induced by administration of a controlcomposition comprising the HCV E1/E2 heterodimer (or HCV E2 polypeptide,or HCV E1 polypeptide) but lacking the CDN, or compared to the number ofHCV E1/E2-specific CD4⁺ T cells and CD8⁺ T cells in the individualbefore administration of the immunogenic composition.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces helper Tlymphocytes (e.g., CD4⁺ T cells) specific for HCV, where the number ofHCV-specific helper T lymphocytes induced is at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 7.5-fold, at least 10-fold, at least 20-fold, at least50-fold, or at least 100-fold, or more than 100-fold, higher than thenumber of HCV-specific helper T cells induced by administration of acontrol composition comprising the HCV E1/E2 heterodimer (or HCV E2polypeptide, or HCV E1 polypeptide) but lacking the CDN, or compared tothe number of HCV-specific CD4⁺ T cells in the individual beforeadministration of the immunogenic composition.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces antibodyspecific for HCV, where the level of HCV-specific antibody induced is atleast at high as the level of HCV-specific antibody induced byadministration of a control composition comprising the HCV E1/E2heterodimer (or HCV E2 polypeptide, or HCV E1 polypeptide) but lackingthe CDN.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces antibodyspecific for HCV, where the level of HCV-specific antibody induced is atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, higher than the level of HCV-specific antibody induced byadministration of a control composition comprising the HCV E1/E2heterodimer (or HCV E2 polypeptide, or HCV E1 polypeptide) but lackingthe CDN, or compared the level of HCV-specific antibody in theindividual before administration of the immunogenic composition.

An immunogenic composition of the present disclosure, when administeredto an individual in need thereof, induces an immune response (e.g., acellular immune response) in the individual to one or more HCVgenotypes. In some cases, an immunogenic composition of the presentdisclosure, when administered to an individual in need thereof, inducesan immune response in the individual to HCV genotype 1. In some cases,an immunogenic composition of the present disclosure, when administeredto an individual in need thereof, induces an immune response in theindividual to HCV genotype 2. In some cases, an immunogenic compositionof the present disclosure when administered to an individual in needthereof, induces an immune response in the individual to HCV genotype 3.In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 1 and HCV genotype 3. In somecases, an immunogenic composition of the present disclosure, whenadministered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 1, HCV genotype 2, and HCVgenotype 3. In some cases, an immunogenic composition of the presentdisclosure, when administered to an individual in need thereof, inducesan immune response in the individual to HCV genotype 1, HCV genotype 2,HCV genotype 3, and HCV genotype 7. In some cases, an immunogeniccomposition of the present disclosure, when administered to anindividual in need thereof, induces an immune response in the individualto HCV genotype 1, HCV genotype 2, HCV genotype 3, HCB genotype 4, HCVgenotype 5, HCV genotype 6, and HCV genotype 7.

HCV E1/E2 Heterodimers; HCV E2 Polypeptides; HCV E1 Polypeptides

HCV E1/E2 heterodimers suitable for use in an immunogenic composition ofthe present disclosure include HCV E1/E2 heterodimers comprisingwild-type HCV E1 polypeptides; HCV E1/E2 heterodimers comprisingwild-type HCV E2 polypeptides; HCV E1/E2 heterodimers comprising variantHCV E1 polypeptides; and HCV E1/E2 heterodimers comprising variant HCVE2 polypeptides. HCV E2 polypeptides suitable for use in an immunogeniccomposition of the present disclosure include wild-type E2 polypeptidesand variant E2 polypeptides. HCV E1 polypeptides suitable for use in animmunogenic composition of the present disclosure include wild-type E1polypeptides and variant E1 polypeptides.

E2 Polypeptides

An E2 polypeptide suitable for inclusion in an E1/E2 heterodimer forinclusion in an immunogenic composition of the present disclosure, orfor inclusion by itself in an immunogenic composition of the presentdisclosure, can have a length of from about 200 amino acids (aa) toabout 250 aa, from about 250 aa to about 275 aa, from about 275 aa toabout 300 aa, from about 300 aa to about 325 aa, from about 325 aa toabout 350 aa, or from about 350 aa to about 365 aa. In some cases, anHCV E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure is an HCV E2 ectodomain polypeptide. In somecases, an HCV E2 polypeptide suitable for inclusion in an immunogeniccomposition of the present disclosure is a full-length HCV E2polypeptide.

In FIG. 1A-AC, the amino acid sequence of E2 is amino acid 384 to aminoacid 746. In FIG. 2A-2B, the amino acid sequence of E2 is amino acid 384to amino acid 751. In FIG. 3A-3C, the amino acid sequence of E2 is aminoacid 385 to amino acid 754. In FIG. 4A-4B, the amino acid sequence of E2is amino acid 384 to amino acid 750. As used herein, an “E2 polypeptide”includes a precursor E2 protein, including the signal sequence; includesa mature E2 polypeptide which lacks this sequence; and includes an E2polypeptide with a heterologous signal sequence. An E2 polypeptide caninclude a C-terminal membrane anchor sequence which occurs atapproximately amino acid positions 715-730 and may extend as far asapproximately amino acid residue 746 (see, Lin et al., J. Virol. (1994)68:5063-5073).

In some cases, a E2 polypeptide suitable for inclusion in an immunogeniccomposition of the present disclosure lacks a portion of its C-terminalregion, e.g., from about amino acid 715 to the C-terminus; from aboutamino acid 625 to the C-terminus; from about amino acid 661 to theC-terminus; from about amino acid 655 to the C-terminus; from aboutamino acid 500 to the C-terminus, where the amino acid numbering is withreference to the numbering in FIG. 1A-1C. See, e.g., U.S. Pat. No.6,521,423.

An E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG. 4A-4B. An E2 polypeptide suitablefor inclusion in an immunogenic composition of the present disclosurecan comprise an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, or at least about 75%, amino acid sequence identity to anamino acid sequence of an E2 polypeptide depicted in FIG. 1A-1C, FIG.2A-2C, FIG. 3A-3C, or FIG. 4A-4B.

An E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.1A-1C. For example, an E2 polypeptide of genotype 1 can comprise anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 384-746 of an aminoacid sequence depicted in FIG. 1A-1C. For example, an E2 polypeptide ofgenotype 1A can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 384-746 of an amino acid sequence identified as 1A and depicted inFIG. 1A-1C. For example, an E2 polypeptide of genotype 1B can comprisean amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 384-746 of an aminoacid sequence identified as 1B and depicted in FIG. 1A-1C. For example,an E2 polypeptide of genotype 1C can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 384-746 of an amino acid sequenceidentified as 1C and depicted in FIG. 1A-1C.

An E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.2A-2C. For example, an E2 polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 384-751 of an amino acid sequencedepicted in FIG. 2A-2C. For example, an E2 polypeptide of genotype 2Acan comprise an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 384-751of the “consensus” amino acid sequence depicted in FIG. 2A-2C. Forexample, an E2 polypeptide of genotype 2B can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 384-751 of the “consensus” aminoacid sequence depicted in FIG. 2A-2C.

An E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.3A-3C. For example, an E2 polypeptide of genotype 3 can comprise anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 385-754 of an aminoacid sequence depicted in FIG. 3A-3C. For example, an E2 polypeptide ofgenotype 3A can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 385-754 of an amino acid sequence identified as 3A and depicted inFIG. 3A-3C. For example, an E2 polypeptide of genotype 3B can comprisean amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 385-754 of the aminoacid sequence identified as 3B and depicted in FIG. 3A-3C. For example,an E2 polypeptide of genotype 3K can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 385-754 of the amino acid sequenceidentified as 3K and depicted in FIG. 3A-3C.

An E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the amino acid sequence of the E2 polypeptide depicted inFIG. 4A-4B. For example, an E2 polypeptide of genotype 7A can comprisean amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 384-750 of the aminoacid sequence depicted in FIG. 4A-4B.

E1 Polypeptides

An HCV E1 polypeptide suitable for inclusion in an E1/E2 heterodimer forinclusion in an immunogenic composition of the present disclosure, orfor inclusion by itself in an immunogenic composition of the presentdisclosure, can have a length of from about 100 amino acids (aa) toabout 150 aa, from about 150 aa to about 175 aa, from about 175 aa toabout 195 aa, from about 131 aa to about 175 aa, or from about 175 aa toabout 193 aa. In some cases, an HCV E1 polypeptide suitable forinclusion in an E1/E2 heterodimer present in an immunogenic compositionof the present disclosure is an HCV E1 ectodomain polypeptide. In somecases, an HCV E1 polypeptide suitable for inclusion in an E1/E2heterodimer present in an immunogenic composition of the presentdisclosure is a full-length HCV E1 polypeptide.

In FIG. 1A-1C, the amino acid sequence of E1 is amino acid 192 to aminoacid 383. In FIG. 2A-2C, the amino acid sequence of E1 is amino acid 192to amino acid 383. In FIG. 3A-3C, the amino acid sequence of E1 is aminoacid 192 to amino acid 384. In FIG. 4A-4B, the amino acid sequence of E1is amino acid 192 to amino acid 383. Amino acids at around 170 throughapproximately 191 serve as a signal sequence for E1. As used herein, “E1polypeptide” includes a precursor E1 protein, including the signalsequence; includes a mature E1 polypeptide which lacks this sequence;and includes an E1 polypeptide with a heterologous signal sequence. AnE1 polypeptide can include a C-terminal membrane anchor sequence whichoccurs at approximately amino acid positions 360-383 (see, e.g., WO96/04301). In some cases, a suitable E1 polypeptide lacks a C-terminalportion that includes a transmembrane region. For example, in somecases, a suitable E1 polypeptide lacks the C-terminal portion from aminoacid 330 to amino acid 384, or from amino acid 360 to amino acid 384. E1polypeptides can be an E1 polypeptide of any genotype, subtype orisolate of HCV. E1 polypeptides of genotype 1 and E1 polypeptides ofgenotype 3 are included in an E1/E2 heterodimer of the presentdisclosure.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 1A-1C, FIG.2A-2C, FIG. 3A-3C, or FIG. 4A-4B.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 1A-1C. Forexample, an E1 polypeptide of genotype 1A can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 192-383 of an amino acid sequenceidentified as 1A and depicted in FIG. 1A-1C. For example, an E1polypeptide of genotype 1B can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 192-383 of an amino acid sequence identified as1B and depicted in FIG. 1A-1C. For example, an E1 polypeptide ofgenotype 1C can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 192-383 of an amino acid sequence identified as 1C and depicted inFIG. 1A-1C.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 2A-2C. Forexample, an E1 polypeptide of genotype 2A can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 192-383 of an amino acid sequenceidentified as 2A and depicted in FIG. 2A-2C. For example, an E1polypeptide of genotype 2B can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 192-383 of an amino acid sequence identified as2B and depicted in FIG. 2A-2C.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity tothe consensus E1 polypeptide amino acid sequence depicted in FIG. 3A-3C.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 4A-4B. Forexample, an E1 polypeptide of genotype 7A can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 192-383 of the amino acid sequencedepicted in FIGS. 4A-4B.

HCV E1 and E2 Polypeptides Comprising Amino Acids from a ProteolyticallyCleavable Linker

As described in more detail below, an HCV E1/E2 heterodimer can begenerated using a method that involves an HCV E1 or an HCV E2polypeptide comprising a heterologous proteolytically cleavable linker.Following enzymatic cleavage of the proteolytically cleavable linker,from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) heterologous amino acids canremain on the HCV E1 or E2 polypeptide. For example, from 1 to 6 (e.g.,1, 2, 3, 4, 5, or 6) heterologous amino acids can remain at theN-terminus of an HCV E2 polypeptide. As another example, from 1 to 6(e.g., 1, 2, 3, 4, 5, or 6) heterologous amino acids can remain at theC-terminus of an HCV E2 polypeptide. As another example, from 1 to 6(e.g., 1, 2, 3, 4, 5, or 6) heterologous amino acids can remain at theN-terminus of an HCV E1 polypeptide. As another example, from 1 to 6(e.g., 1, 2, 3, 4, 5, or 6) heterologous amino acids can remain at theC-terminus of an HCV E1 polypeptide.

In some cases, amino acids C-terminal to the proteolytic cleavage sitein a proteolytically cleavable linker are Gly-Pro, Ser, Gly, or Gly-Ser.Thus, in some cases, a modified HCV E1 polypeptide comprises, appendedto the N-terminus of an HCV E1 polypeptide: Gly-Pro, Ser, Gly, orGly-Ser. In other words, in some cases, a modified HCV E1 polypeptidecomprises, in order from N-terminus to C-terminus: a) Gly-Pro, Ser, Gly,or Gly-Ser; and b) an HCV E1 polypeptide.

In some cases, amino acids C-terminal to the proteolytic cleavage sitein a proteolytically cleavable linker are Gly-Pro, Ser, Gly, or Gly-Ser.Thus, in some cases, a modified HCV E2 polypeptide comprises, appendedto the N-terminus of an HCV E2 polypeptide: Gly-Pro, Ser, Gly, orGly-Ser. In other words, in some cases, a modified HCV E2 polypeptidecomprises, in order from N-terminus to C-terminus: a) Gly-Pro, Ser, Gly,or Gly-Ser; and b) an HCV E2 polypeptide.

In some cases, amino acids N-terminal to the proteolytic cleavage sitein a proteolytically cleavable linker are LEVLFQ (SEQ ID NO:122),ENLYYFQ (SEQ ID NO:150), LVPR (SEQ ID NO:124), I(E/D)GR (SEQ ID NO:125),or DDDDK (SEQ ID NO:123). Thus, in some cases, a modified HCV E1polypeptide comprises, appended to the C-terminus of an HCV E1polypeptide: LEVLFQ (SEQ ID NO:122), ENLYYFQ (SEQ ID NO:150), LVPR (SEQID NO:124), I(E/D)GR (SEQ ID NO:125), or DDDDK (SEQ ID NO:123). In otherwords, in some cases, a modified HCV E1 polypeptide comprises, in orderfrom N-terminus to C-terminus: a) an HCV E1 polypeptide; and b) LEVLFQ(SEQ ID NO:122), ENLYYFQ (SEQ ID NO:150), LVPR (SEQ ID NO:124), I(E/D)GR(SEQ ID NO:125), or DDDDK (SEQ ID NO:123).

In some cases, amino acids N-terminal to the proteolytic cleavage sitein a proteolytically cleavable linker are LEVLFQ (SEQ ID NO:122),ENLYYFQ (SEQ ID NO:150), LVPR (SEQ ID NO:124), I(E/D)GR (SEQ ID NO:125),or DDDDK (SEQ ID NO:123). Thus, in some cases, a modified HCV E2polypeptide comprises, appended to the C-terminus of an HCV E2polypeptide: LEVLFQ (SEQ ID NO:122), ENLYYFQ (SEQ ID NO:150), LVPR (SEQID NO:124), I(E/D)GR (SEQ ID NO:125), or DDDDK (SEQ ID NO:123). In otherwords, in some cases, a modified HCV E2 polypeptide comprises, in orderfrom N-terminus to C-terminus: a) an HCV E2 polypeptide; and b) LEVLFQ(SEQ ID NO:122), ENLYYFQ (SEQ ID NO:150), LVPR (SEQ ID NO:124), I(E/D)GR(SEQ ID NO:125), or DDDDK (SEQ ID NO:123).

In some cases, a flexible linker of from 1 to 10 amino acids isinterposed between the proteolytically cleavable linker and the HCV E1or E2 polypeptide. Flexible linkers are intrinsically disorderedflexible linker domains or loops that vary in length and can be rich inpolar uncharged amino acids. Flexible linkers include, e.g., glycinepolymers (G)_(n), glycine-serine polymers (including, for example,(GS)_(n), GSGGS_(n) (SEQ ID NO:108), GGSGGS_(n) (SEQ ID NO:109), andGGGS_(n) (SEQ ID NO:110), where n is an integer of at least one, e.g.,where n is 1, 2, 3, 4, 5, or 6); glycine-alanine polymers, such asGAGAGAGA and the like; and alanine-serine polymers, e.g., SASASASA andthe like. Exemplary linkers can comprise amino acid sequences including,but not limited to, GGSG (SEQ ID NO:111), GGSGG (SEQ ID NO:112), GSGSG(SEQ ID NO:113), GSGGG (SEQ ID NO:114), GGGSG (SEQ ID NO:115), GSSSG(SEQ ID NO:116), and the like.

For example, in some cases, a modified E1 polypeptide comprises, inorder from N-terminus to C-terminus: a) Gly-Pro, Ser, Gly, or Gly-Ser;b) a flexible linker of from 1 to 10 amino acids; and c) an HCV E1polypeptide.

As another example, in some cases, a modified E2 polypeptide comprises,in order from N-terminus to C-terminus: a) Gly-Pro, Ser, Gly, orGly-Ser; b) a flexible linker of from 1 to 10 amino acids; and c) an HCVE2 polypeptide.

As another in some cases, a modified E1 polypeptide comprises, fromN-terminus to C-terminus: a) an HCV E1 polypeptide; b) a flexible linkerof from 1 to 10 amino acids; and c) LEVLFQ (SEQ ID NO:122), ENLYYFQ (SEQID NO:150), LVPR (SEQ ID NO:124), I(E/D)GR (SEQ ID NO:125), or DDDDK(SEQ ID NO:123).

As another in some cases, a modified E2 polypeptide comprises, fromN-terminus to C-terminus: a) an HCV E2 polypeptide; b) a flexible linkerof from 1 to 10 amino acids; and c) LEVLFQ (SEQ ID NO:122), ENLYYFQ (SEQID NO:150), LVPR (SEQ ID NO:124), I(E/D)GR (SEQ ID NO:125), or DDDDK(SEQ ID NO:123).

E2 with N-Terminal Heterologous Amino Acids

In some cases, an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises a modifiedHCV E2 polypeptide with from 1 to 6 amino acids from the proteolyticallycleavable linker on the N-terminus of the E2 polypeptide. In some cases,an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises: a) an HCV E1polypeptide; and b) a modified E2 polypeptide comprising, in order fromN-terminus to C-terminus: i) from 1 to 6 heterologous amino acidswherein the from 1 to 6 heterologous amino acids are C-terminal to asite of proteolytic cleavage in a proteolytically cleavable linker; andii) an HCV E2 polypeptide.

Proteolytically cleavable linkers are described elsewhere herein.Following proteolytic cleavage of a precursor polypeptide, as describedherein, a modified E2 polypeptide is generated, which modified E2polypeptide comprises, at its N-terminus, amino acids C-terminal to theprotease cleavage site within the proteolytically cleavable linker.

For example, where the proteolytically cleavable linker comprises aPreScission cleavage site (LEVLFQGP; SEQ ID NO:117), where cleavageoccurs between the glutamine and the glycine, a modified E2 polypeptidepresent in a heterodimeric E1/E2 polypeptide suitable for inclusion inan immunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) Gly-Pro; and b) an HCV E2 polypeptide.As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQS; SEQ ID NO:118), where cleavage occursbetween the glutamine and the serine, a modified E2 polypeptide presentin a heterodimeric E1/E2 polypeptide suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) Ser; and b) an HCV E2 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQG; SEQ ID NO:119), where cleavage occursbetween the glutamine and the glycine, a modified E2 polypeptide presentin a heterodimeric E1/E2 polypeptide suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) Gly; and b) an HCV E2 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa thrombin cleavage site (LVPRGS; SEQ ID NO:120), where cleavage occursbetween the arginine and the glycine, a modified E2 polypeptide presentin a heterodimeric E1/E2 polypeptide suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) Gly-Ser; and an HCV E2 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa Factor Xa cleavage site (I(E/D)GRX, where X is any amino acid exceptarginine or proline; SEQ ID NO:121), where cleavage occurs between thearginine and the X, a modified E2 polypeptide present in a heterodimericE1/E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure comprises, in order from N-terminus toC-terminus: a) X (where X is any amino acid except arginine or proline);and an HCV E2 polypeptide.

Thus, for example, in some cases, an HCV E1/E2 heterodimer suitable forinclusion in an immunogenic composition of the present disclosurecomprises: a) an HCV E1 polypeptide; and b) a modified E2 polypeptidecomprising, in order from N-terminus to C-terminus: i) from 1 to 6heterologous amino acids, wherein the from 1 to 6 (e.g., 1, 2, 3, 4, 5,or 6) heterologous amino acids are C-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; and ii) an HCV E2polypeptide. In some cases, the 1 to 6 heterologous amino acids areGly-Pro. In some cases, the 1 to 6 heterologous amino acids is Ser. Insome cases, the 1 to 6 heterologous amino acids is Gly. In some cases,the 1 to 6 heterologous amino acids are Gly-Ser.

As another example, in some cases, an HCV E1/E2 heterodimer suitable forinclusion in an immunogenic composition of the present disclosurecomprises: a) an HCV E2 polypeptide; and b) a modified E1 polypeptidecomprising, in order from N-terminus to C-terminus: i) from 1 to 6heterologous amino acids, wherein the from 1 to 6 (e.g., 1, 2, 3, 4, 5,or 6) heterologous amino acids are C-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; and ii) an HCV E1polypeptide. In some cases, the 1 to 6 heterologous amino acids areGly-Pro. In some cases, the 1 to 6 heterologous amino acids is Ser. Insome cases, the 1 to 6 heterologous amino acids is Gly. In some cases,the 1 to 6 heterologous amino acids are Gly-Ser.

E1 with N-Terminal Heterologous Amino Acids

In some cases, an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises a modifiedHCV E1 polypeptide with from 1 to 6 amino acids from a proteolyticallycleavable linker on the N-terminus of the E1 polypeptide. In some cases,an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises: a) an HCV E2polypeptide; and b) a modified E1 polypeptide comprising, in order fromN-terminus to C-terminus: i) from 1 to 6 heterologous amino acidswherein the from 1 to 6 heterologous amino acids are C-terminal to asite of proteolytic cleavage in a proteolytically cleavable linker; andii) an HCV E1 polypeptide.

Proteolytically cleavable linkers are described elsewhere herein.Following proteolytic cleavage of a precursor polypeptide (e.g., aprecursor polypeptide comprising, in order from N-terminus toC-terminus: a) an Fc polypeptide or an HCV E2 polypeptide; b) aproteolytically cleavable linker; and c) an HCV E1 polypeptide), amodified E1 polypeptide is generated, which modified E1 polypeptidecomprises, at its N-terminus, amino acids C-terminal to the proteasecleavage site within the proteolytically cleavable linker.

For example, where the proteolytically cleavable linker comprises aPreScission cleavage site (LEVLFQGP; SEQ ID NO:117), where cleavageoccurs between the glutamine and the glycine, a modified E1 polypeptidepresent in an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) Gly-Pro; and b) an HCV E1 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQS; SEQ ID NO:118), where cleavage occursbetween the glutamine and the serine, a modified E1 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) Ser; and b) an HCV E1 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQG; SEQ ID NO:119), where cleavage occursbetween the glutamine and the glycine, a modified E1 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) Gly; and b) an HCV E1 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa thrombin cleavage site (LVPRGS; SEQ ID NO:120), where cleavage occursbetween the arginine and the glycine, a modified E1 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) Gly-Ser; and an HCV E1 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa Factor Xa cleavage site (I(E/D)GRX, where X is any amino acid exceptarginine or proline; SEQ ID NO:121), where cleavage occurs between thearginine and the X, a modified E1 polypeptide present in an HCV E1/E2heterodimer suitable for inclusion in an immunogenic composition of thepresent disclosure comprises, in order from N-terminus to C-terminus: a)X (where X is any amino acid except arginine or proline); and an HCV E1polypeptide.

E2 with C-Terminal Heterologous Amino Acids

In some cases, an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises a modifiedHCV E2 polypeptide with from 1 to 6 amino acids from a proteolyticallycleavable linker on the C-terminus of the E2 polypeptide. In some cases,an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises: a) an HCV E1polypeptide; and b) a modified E2 polypeptide comprising, in order fromN-terminus to C-terminus: i) an HCV E2 polypeptide; and ii) from 1 to 6heterologous amino acids wherein the from 1 to 6 heterologous aminoacids are N-terminal to a site of proteolytic cleavage in aproteolytically cleavable linker.

Proteolytically cleavable linkers are described elsewhere herein.Following proteolytic cleavage of a precursor polypeptide (e.g., aprecursor polypeptide comprising, in order from N-terminus toC-terminus: a) HCV E2 polypeptide; b) a proteolytically cleavablelinker; and c) an Fc polypeptide or an HCV E1 polypeptide), a modifiedE2 polypeptide is generated, which modified E2 polypeptide comprises, atits C-terminus, amino acids N-terminal to the protease cleavage sitewithin the proteolytically cleavable linker.

For example, where the proteolytically cleavable linker comprises aPreScission cleavage site (LEVLFQGP; SEQ ID NO:117), where cleavageoccurs between the glutamine and the glycine, a modified E2 polypeptidepresent in an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an HCV E2 polypeptide; and b) LEVLFQ(SEQ ID NO:122).

As another example, where the proteolytically cleavable linker comprisesan enterokinase cleavage site (DDDDK; SEQ ID NO:123), where cleavageoccurs C-terminal to the Lys, a modified E2 polypeptide present in anHCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E2 polypeptide; and b) DDDDK (SEQ IDNO:123).

As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQG; SEQ ID NO:119), where cleavage occursbetween the glutamine and the glycine, a modified E2 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E2 polypeptide; and b) ENLYFQ (SEQID NO:151).

As another example, where the proteolytically cleavable linker comprisesa thrombin cleavage site (LVPRGS; SEQ ID NO:120), where cleavage occursbetween the arginine and the glycine, a modified E2 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E2 polypeptide; and LVPR (SEQ IDNO:124).

As another example, where the proteolytically cleavable linker comprisesa Factor Xa cleavage site (I(E/D)GRX, where X is any amino acid exceptarginine or proline; SEQ ID NO:121), where cleavage occurs between thearginine and the X, a modified E2 polypeptide present in an HCV E1/E2heterodimer suitable for inclusion in an immunogenic composition of thepresent disclosure comprises, in order from N-terminus to C-terminus: a)an HCV E2 polypeptide; and I(E/D)GR (SEQ ID NO:125).

E1 with C-Terminal Heterologous Amino Acids

In some cases, an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises a modifiedHCV E1 polypeptide with from 1 to 6 amino acids from a proteolyticallycleavable linker on the C-terminus of the E1 polypeptide. In some cases,an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises: a) an HCV E2polypeptide; and b) a modified E1 polypeptide comprising, in order fromN-terminus to C-terminus: i) an HCV E1 polypeptide; and ii) from 1 to 6heterologous amino acids wherein the from 1 to 6 heterologous aminoacids are N-terminal to a site of proteolytic cleavage in aproteolytically cleavable linker.

Proteolytically cleavable linkers are described elsewhere herein.Following proteolytic cleavage of a precursor polypeptide (e.g., aprecursor polypeptide comprising, in order from N-terminus toC-terminus: a) HCV E1 polypeptide; b) a proteolytically cleavablelinker; and c) an Fc polypeptide or an HCV E2 polypeptide), a modifiedE1 polypeptide is generated, which modified E1 polypeptide comprises, atits C-terminus, amino acids N-terminal to the protease cleavage sitewithin the proteolytically cleavable linker.

For example, where the proteolytically cleavable linker comprises aPreScission cleavage site (LEVLFQGP; SEQ ID NO:117), where cleavageoccurs between the glutamine and the glycine, a modified E1 polypeptidepresent in an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an HCV E1 polypeptide; and b) LEVLFQ(SEQ ID NO:122).

As another example, where the proteolytically cleavable linker comprisesan enterokinase cleavage site (DDDDK; SEQ ID NO:123), where cleavageoccurs C-terminal to the Lys, a modified E1 polypeptide present in anHCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E1 polypeptide; and b) DDDDK (SEQ IDNO:123).

As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQG; SEQ ID NO:119), where cleavage occursbetween the glutamine and the glycine, a modified E1 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E1 polypeptide; and b) ENLYFQ (SEQID NO:151).

As another example, where the proteolytically cleavable linker comprisesa thrombin cleavage site (LVPRGS; SEQ ID NO:120), where cleavage occursbetween the arginine and the glycine, a modified E1 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E1 polypeptide; and LVPR (SEQ IDNO:124).

As another example, where the proteolytically cleavable linker comprisesa Factor Xa cleavage site (I(E/D)GRX, where X is any amino acid exceptarginine or proline; SEQ ID NO:121), where cleavage occurs between thearginine and the X, a modified E1 polypeptide present in an HCV E1/E2heterodimer suitable for inclusion in an immunogenic composition of thepresent disclosure comprises, in order from N-terminus to C-terminus: a)an HCV E1 polypeptide; and I(E/D)GR (SEQ ID NO:125).

Additional Polypeptides

In any of the above-described embodiments, one or both of thepolypeptide chains of the E1/E2 heterodimer can include one or moreadditional polypeptides. For example, the E1 polypeptide, the E2polypeptide, or both the E1 and the E2 polypeptide, can include anaffinity tag. Suitable affinity tags include, e.g., immunoglobulin Fcpolypeptides, a poly(histidine) tag (e.g., His6), a maltose bindingprotein (MBP), a glutathione-S-transferase (GST) polypeptide,calmodulin-binding peptide (CBP), Streptavidin-binding peptide (SBP),Strep-tag II, FLAG (e.g., DYKDDDDK (SEQ ID NO:126), hemagglutinin (HA)(e.g., YPYDVPDYA (SEQ ID NO:127), c-myc T7 ((e.g., EQKLISEEDL; SEQ IDNO:128), Glu-Glu, starch-binding domain (SBD), and Flag-Acidic-TargetTag (FATT), and the like.

In some cases, an E1/E2 heterodimer included in a composition of thepresent disclosure includes a variant E2 polypeptide. In some cases, theE1 polypeptide or the variant E2 polypeptide can include an Ig Fcpolypeptide at the C-terminus of the E1 polypeptide or the variant E2polypeptide. As another example, in some cases, the E1 polypeptide orthe variant E2 polypeptide can include an Ig Fc polypeptide at theN-terminus of the E1 polypeptide or the variant E2 polypeptide. Ig Fcpolypeptides are known in the art, and are described elsewhere herein.

T-Cell Epitope Polypeptides

In some cases, one or both of the polypeptide chains of the E1/E2heterodimer present in an immunogenic composition of the presentdisclosure can include a T-cell epitope polypeptide. In some cases, anE2 polypeptide present in an E1/E2 heterodimer present in an immunogeniccomposition of the present disclosure includes a T-cell epitopepolypeptide. In some cases, an E1 polypeptide present in an E1/E2heterodimer present in an immunogenic composition of the presentdisclosure includes a T-cell epitope polypeptide. In some cases, an HCVE2 polypeptide present in an immunogenic composition of the presentdisclosure includes a T-cell epitope polypeptide. In some cases, an HCVE1 polypeptide present in an immunogenic composition of the presentdisclosure includes a T-cell epitope polypeptide. In these embodiments,the T-cell epitope is covalently linked to the E1 and/or E2 polypeptide.For example, in some cases, the T-cell epitope is covalently linked tothe amino terminus (N-terminus) of the HCV E1 polypeptide. In somecases, the T-cell epitope is covalently linked to the carboxyl terminus(C-terminus) of the HCV E1 polypeptide. Thus, e.g., in some cases, animmunogenic composition of the present disclosure comprises: a) a fusionpolypeptide comprising, in order from N-terminus to C-terminus: i) anHCV E1 polypeptide; and ii) a T-cell epitope polypeptide; and b) a CDN.In some cases, an immunogenic composition of the present disclosurecomprises: a) a fusion polypeptide comprising, in order from N-terminusto C-terminus: i) an HCV E2 polypeptide; and ii) a T-cell epitopepolypeptide; and b) a CDN. In some cases, an immunogenic composition ofthe present disclosure comprises: a) a fusion polypeptide comprising, inorder from N-terminus to C-terminus: i) a T-cell epitope polypeptide;and ii) an HCV E1 polypeptide; and b) a CDN. In some cases, animmunogenic composition of the present disclosure comprises: a) a fusionpolypeptide comprising, in order from N-terminus to C-terminus: i) aT-cell epitope polypeptide; and ii) an HCV E2 polypeptide; and b) a CDN.In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1E2 heterodimer comprising i) a fusion polypeptidecomprising, in order from N-terminus to C-terminus: an HCV E1polypeptide; and a T-cell epitope polypeptide; and ii) an HCV E2polypeptide; and b) a CDN. In some cases, an immunogenic composition ofthe present disclosure comprises: a) an HCV E1E2 heterodimer comprisingi) a fusion polypeptide comprising, in order from N-terminus toC-terminus: an HCV E2 polypeptide; and a T-cell epitope polypeptide; andii) an HCV E2 polypeptide; and b) a CDN. In some cases, an immunogeniccomposition of the present disclosure comprises: a) an HCV E1E2heterodimer comprising i) a fusion polypeptide comprising, in order fromN-terminus to C-terminus: a T-cell epitope polypeptide; and an HCV E1polypeptide; and ii) an HCV E2 polypeptide; and b) a CDN. In some cases,an immunogenic composition of the present disclosure comprises: a) anHCV E1E2 heterodimer comprising i) a fusion polypeptide comprising, inorder from N-terminus to C-terminus: a T-cell epitope polypeptide; andan HCV E2 polypeptide; and ii) an HCV E1 polypeptide; and b) a CDN.

In some cases, an immunogenic composition of the present disclosurecomprises a T-cell epitope polypeptide, where the T-cell epitopepolypeptide is not covalently linked to the HCV E1/E2 heterodimer, theHCV E1 polypeptide or the HCV E2 polypeptide. For example, in somecases, an immunogenic composition of the present disclosure comprises:a) an HCV E1/E2 heterodimer; b) a CDN; and c) a T-cell epitopepolypeptide. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E2 polypeptide; b) a CDN; and c) aT-cell epitope polypeptide. In some cases, an immunogenic composition ofthe present disclosure comprises: a) an HCV E1 polypeptide; b) a CDN;and c) a T-cell epitope polypeptide.

A T-cell epitope polypeptide suitable for inclusion in an immunogeniccomposition of the present disclosure comprises a T-cell epitope presentin an HCV protein other than E1 and E2. T-cell epitope polypeptidessuitable for inclusion in an immunogenic composition of the presentdisclosure comprise T cell epitopes that are conserved among differentHCV genotypes leading to cross-reactive cellular immune responses. Insome cases, the T-cell epitope polypeptide does not include a neotope;for example, in some cases, the T-cell epitope polypeptide does notinclude a junction formed by amino acid sequences that do not naturallyoccur adjacent to one another in a naturally-occurring HCV polypeptide.

In some cases, the T-cell epitope polypeptide comprises 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, or more than 10 (e.g., from 10 to 15, from 15 to 20,from 20 to 25, or from 25 to 30, or more than 30), T cell epitopes.T-cell epitopes are epitopes that, when presented with a majorhistocompatibility complex (MHC) (e.g., a human leukocyte antigen (HLA))Class I or MHC Class II molecule, are recognized and bound by a T-cellreceptor (TCR) present on a T cell surface. T-cell epitopes includeepitopes recognized by cytotoxic T cells (e.g., CD8⁺ T cells), andepitopes recognized by helper T cells (e.g., CD4⁺ T cells).

The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) anHCV NS4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5Apolypeptide; f) an HCV NS5B polypeptide; g) an HCV core polypeptide; orh) an HCV p7 polypeptide. In some cases, the one or more T-cell epitopesare T-cell epitopes present in an HCV NS3 polypeptide. In some cases,the T-cell epitope polypeptide further comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) ameningococcal outer membrane protein. A suitable source of T-cellepitopes includes non-toxic mutants of toxins, where the mutants arereferred to as “cross-reactive material (CRM).” Other examples of strongT helper epitopes are diphtheria toxoid, tetanus toxoid, meningococcalouter membrane protein, or mutant diphtheria protein CRM197 (see, e.g.:world-wide-website: medscape.com/viewarticle/431127).

In some cases, the T-cell epitope polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS3 T-cell epitope. In some cases, the T-cell epitope polypeptidecomprises 2 or more T-cell epitopes. In some cases, the T-cell epitopepolypeptide comprises 2 or more HCV-NS3 T-cell epitopes. In some cases,the T-cell epitope polypeptide comprises 3 or more HCV-NS3 T-cellepitopes. In some cases, the T-cell epitope polypeptide comprises 4 ormore HCV-NS3 T-cell epitopes. In some cases, the T-cell epitopepolypeptide comprises a single HCV-NS3 CD4⁺ T-cell epitope. In somecases, the T-cell epitope polypeptide comprises 2 or more HCV-NS3 CD4⁺T-cell epitopes. In some cases, the T-cell epitope polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the T-cell epitopepolypeptide comprises a single HCV-NS3 CD8⁺ T-cell epitope. In somecases, the T-cell epitope polypeptide comprises 2 or more HCV-NS3 CD8⁺T-cell epitopes. In some cases, the T-cell epitope polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the T-cell epitope polypeptide comprises atleast one HCV-NS3 CD4⁺ T cell epitope and at least one HCV-NS3 CD8⁺ Tcell epitope. In some cases, T-cell epitope polypeptide comprises 2 ormore HCV-NS3 CD4⁺ T-cell epitopes and 2 or more HCV-NS3 CD8⁺ T-cellepitopes. In some cases, the T-cell epitope polypeptide comprises 2, 3,4, or 5 HCV-NS3 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS3 CD8⁺T-cell epitopes.

In some cases, the T-cell epitope polypeptide comprises a single T-cellepitope. In some cases, the T-cell epitope polypeptide comprises asingle HCV-NS2 T-cell epitope. In some cases, the T-cell epitopepolypeptide comprises 2 or more T-cell epitopes. In some cases, theT-cell epitope polypeptide comprises 2 or more HCV-NS2 T-cell epitopes.In some cases, the T-cell epitope polypeptide comprises 3 or moreHCV-NS2 T-cell epitopes. In some cases, the T-cell epitope polypeptidecomprises 4 or more HCV-NS2 T-cell epitopes. In some cases, the T-cellepitope polypeptide comprises a single HCV-NS2 CD4⁺ T-cell epitope. Insome cases, the T-cell epitope polypeptide comprises 2 or more HCV-NS2CD4⁺ T-cell epitopes. In some cases, the T-cell epitope polypeptidecomprises one or more HCV CD8⁺ T cell epitopes. In some cases, theT-cell epitope polypeptide comprises a single HCV-NS2 CD8⁺ T-cellepitope. In some cases, the T-cell epitope polypeptide comprises 2 ormore HCV-NS2 CD8⁺ T-cell epitopes. In some cases, the T-cell epitopepolypeptide comprises at least one HCV CD4⁺ T cell epitope and at leastone HCV CD8⁺ T cell epitope. In some cases, the T-cell epitopepolypeptide comprises at least one HCV-NS2 CD4⁺ T cell epitope and atleast one HCV-NS2 CD8⁺ T cell epitope. In some cases, T-cell epitopepolypeptide comprises 2 or more HCV-NS2 CD4⁺ T-cell epitopes and 2 ormore HCV-NS2 CD8⁺ T-cell epitopes. In some cases, the T-cell epitopepolypeptide comprises 2, 3, 4, or 5 HCV-NS2 CD4⁺ T-cell epitopes and 2,3, 4, or 5 HCV-NS2 CD8⁺ T-cell epitopes.

In some cases, the T cell epitope polypeptide comprises a single T-cellepitope. In some cases, the T cell epitope polypeptide comprises asingle HCV-NS4A T-cell epitope. In some cases, the T cell epitopepolypeptide comprises 2 or more T-cell epitopes. In some cases, the Tcell epitope polypeptide comprises 2 or more HCV-NS4A T-cell epitopes.In some cases, the T cell epitope polypeptide comprises 3 or moreHCV-NS4A T-cell epitopes. In some cases, the T cell epitope polypeptidecomprises 4 or more HCV-NS4A T-cell epitopes. In some cases, the T cellepitope polypeptide comprises a single HCV-NS4A CD4⁺ T-cell epitope. Insome cases, the T cell epitope polypeptide comprises 2 or more HCV-NS4ACD4⁺ T-cell epitopes. In some cases, the T cell epitope polypeptidecomprises one or more HCV CD8⁺ T cell epitopes. In some cases, the Tcell epitope polypeptide comprises a single HCV-NS4A CD8⁺ T-cellepitope. In some cases, the T cell epitope polypeptide comprises 2 ormore HCV-NS4A CD8⁺ T-cell epitopes. In some cases, the T cell epitopepolypeptide comprises at least one HCV CD4⁺ T cell epitope and at leastone HCV CD8⁺ T cell epitope. In some cases, the T cell epitopepolypeptide comprises at least one HCV-NS4A CD4⁺ T cell epitope and atleast one HCV-NS4A CD8⁺ T cell epitope. In some cases, T cell epitopepolypeptide comprises 2 or more HCV-NS4A CD4⁺ T-cell epitopes and 2 ormore HCV-NS4A CD8⁺ T-cell epitopes. In some cases, the T cell epitopepolypeptide comprises 2, 3, 4, or 5 HCV-NS4A CD4⁺ T-cell epitopes and 2,3, 4, or 5 HCV-NS4A CD8⁺ T-cell epitopes.

In some cases, the T cell epitope polypeptide comprises a single T-cellepitope. In some cases, the T cell epitope polypeptide comprises asingle HCV-NS5A T-cell epitope. In some cases, the T cell epitopepolypeptide comprises 2 or more T-cell epitopes. In some cases, the Tcell epitope polypeptide comprises 2 or more HCV-NS5A T-cell epitopes.In some cases, the T cell epitope polypeptide comprises 3 or moreHCV-NS5A T-cell epitopes. In some cases, the T cell epitope polypeptidecomprises 4 or more HCV-NS5A T-cell epitopes. In some cases, the T cellepitope polypeptide comprises a single HCV-NS5A CD4⁺ T-cell epitope. Insome cases, the T cell epitope polypeptide comprises 2 or more HCV-NS5ACD4⁺ T-cell epitopes. In some cases, the T cell epitope polypeptidecomprises one or more HCV CD8⁺ T cell epitopes. In some cases, the Tcell epitope polypeptide comprises a single HCV-NS5A CD8⁺ T-cellepitope. In some cases, the T cell epitope polypeptide comprises 2 ormore HCV-NS5A CD8⁺ T-cell epitopes. In some cases, the T cell epitopepolypeptide comprises at least one HCV CD4⁺ T cell epitope and at leastone HCV CD8⁺ T cell epitope. In some cases, the T cell epitopepolypeptide comprises at least one HCV-NS5A CD4⁺ T cell epitope and atleast one HCV-NS5A CD8⁺ T cell epitope. In some cases, T cell epitopepolypeptide comprises 2 or more HCV-NS5A CD4⁺ T-cell epitopes and 2 ormore HCV-NS5A CD8⁺ T-cell epitopes. In some cases, the T cell epitopepolypeptide comprises 2, 3, 4, or 5 HCV-NS5A CD4⁺ T-cell epitopes and 2,3, 4, or 5 HCV-NS5A CD8⁺ T-cell epitopes.

In some cases, the T cell epitope polypeptide comprises a single T-cellepitope. In some cases, the T cell epitope polypeptide comprises asingle HCV-NS5B T-cell epitope. In some cases, the T cell epitopepolypeptide comprises 2 or more T-cell epitopes. In some cases, the Tcell epitope polypeptide comprises 2 or more HCV-NS5B T-cell epitopes.In some cases, the T cell epitope polypeptide comprises 3 or moreHCV-NS5B T-cell epitopes. In some cases, the T cell epitope polypeptidecomprises 4 or more HCV-NS5B T-cell epitopes. In some cases, the T cellepitope polypeptide comprises a single HCV-NS5B CD4⁺ T-cell epitope. Insome cases, the T cell epitope polypeptide comprises 2 or more HCV-NS5BCD4⁺ T-cell epitopes. In some cases, the T cell epitope polypeptidecomprises one or more HCV CD8⁺ T cell epitopes. In some cases, the Tcell epitope polypeptide comprises a single HCV-NS5B CD8⁺ T-cellepitope. In some cases, the T cell epitope polypeptide comprises 2 ormore HCV-NS5B CD8⁺ T-cell epitopes. In some cases, the T cell epitopepolypeptide comprises at least one HCV CD4⁺ T cell epitope and at leastone HCV CD8⁺ T cell epitope. In some cases, the T cell epitopepolypeptide comprises at least one HCV-NS5B CD4⁺ T cell epitope and atleast one HCV-NS5B CD8⁺ T cell epitope. In some cases, T cell epitopepolypeptide comprises 2 or more HCV-NS5B CD4⁺ T-cell epitopes and 2 ormore HCV-NS5B CD8⁺ T-cell epitopes. In some cases, the T cell epitopepolypeptide comprises 2, 3, 4, or 5 HCV-NS5B CD4⁺ T-cell epitopes and 2,3, 4, or 5 HCV-NS5B CD8⁺ T-cell epitopes.

In some cases, the T cell epitope polypeptide comprises a single T-cellepitope. In some cases, the T cell epitope polypeptide comprises asingle HCV-core T-cell epitope. In some cases, the T cell epitopepolypeptide comprises 2 or more T-cell epitopes. In some cases, the Tcell epitope polypeptide comprises 2 or more HCV-core T-cell epitopes.In some cases, the T cell epitope polypeptide comprises 3 or moreHCV-core T-cell epitopes. In some cases, the T cell epitope polypeptidecomprises 4 or more HCV-core T-cell epitopes. In some cases, the T cellepitope polypeptide comprises a single HCV-core CD4⁺ T-cell epitope. Insome cases, the T cell epitope polypeptide comprises 2 or more HCV-coreCD4⁺ T-cell epitopes. In some cases, the T cell epitope polypeptidecomprises one or more HCV CD8⁺ T cell epitopes. In some cases, the Tcell epitope polypeptide comprises a single HCV-core CD8⁺ T-cellepitope. In some cases, the T cell epitope polypeptide comprises 2 ormore HCV-core CD8⁺ T-cell epitopes. In some cases, the T cell epitopepolypeptide comprises at least one HCV CD4⁺ T cell epitope and at leastone HCV CD8⁺ T cell epitope. In some cases, the T cell epitopepolypeptide comprises at least one HCV-core CD4⁺ T cell epitope and atleast one HCV-core CD8⁺ T cell epitope. In some cases, T cell epitopepolypeptide comprises 2 or more HCV-core CD4⁺ T-cell epitopes and 2 ormore HCV-core CD8⁺ T-cell epitopes. In some cases, the T cell epitopepolypeptide comprises 2, 3, 4, or 5 HCV-core CD4⁺ T-cell epitopes and 2,3, 4, or 5 HCV-core CD8⁺ T-cell epitopes.

In some cases, the T cell epitope polypeptide comprises a single T-cellepitope. In some cases, the T cell epitope polypeptide comprises asingle HCV-p7 T-cell epitope. In some cases, the T cell epitopepolypeptide comprises 2 or more T-cell epitopes. In some cases, the Tcell epitope polypeptide comprises 2 or more HCV-p7 T-cell epitopes. Insome cases, the T cell epitope polypeptide comprises 3 or more HCV-p7T-cell epitopes. In some cases, the T cell epitope polypeptide comprises4 or more HCV-p7 T-cell epitopes. In some cases, the T cell epitopepolypeptide comprises a single HCV-p7 CD4⁺ T-cell epitope. In somecases, the T cell epitope polypeptide comprises 2 or more HCV-core CD4⁺T-cell epitopes. In some cases, the T cell epitope polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the T cell epitopepolypeptide comprises a single HCV-p7 CD8⁺ T-cell epitope. In somecases, the T cell epitope polypeptide comprises 2 or more HCV-p7 CD8⁺T-cell epitopes. In some cases, the T cell epitope polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the T cell epitope polypeptide comprises atleast one HCV-p7 CD4⁺ T cell epitope and at least one HCV-p7 CD8⁺ T cellepitope. In some cases, T cell epitope polypeptide comprises 2 or moreHCV-p7 CD4⁺ T-cell epitopes and 2 or more HCV-p7 CD8⁺ T-cell epitopes.In some cases, the T-cell epitope polypeptide comprises 2, 3, 4, or 5HCV-p7 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-p7 CD8⁺ T-cellepitopes.

In some cases, the T cell epitope polypeptide comprises 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, or 63, of the T-cell epitopes set out in FIG. 9A-9B. In somecases, the T cell epitope polypeptide comprises from 1 to 3, from 3 to5, from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, or from 25to 30 of the T-cell epitopes set out in FIG. 9A-9B. For example, in somecases, the T cell epitope polypeptide comprises the T-cell epitopesdesignated NS3-3, NS3-4, and NS3-11 in FIG. 9A-9B and FIG. 11A-11N. Asanother example, in some cases, the T cell epitope polypeptide comprisesthe T-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 inFIG. 9A-9B and FIG. 11A-11N. As another example, in some cases, the Tcell epitope polypeptide comprises the T-cell epitopes designated NS3-3,NS3-4, NS3-5, and NS3-11 in FIG. 9A-9B and FIG. 11A-11N. As anotherexample, in some cases, the T cell epitope polypeptide comprises theT-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-11,NS3-12, and NS3-13 in FIG. 9A-9B and FIG. 11A-11N. As another example,in some cases, the T cell epitope polypeptide comprises the T-cellepitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 9A-9B and FIG. 11A-11N. As another example,in some cases, the T cell epitope polypeptide comprises the T-cellepitopes designated Core-1, Core-2, Core-3, Core-4, Core-5, Core-6,Core-7, Core-8, Core-9, Core-10, Core-11, Core-12, Core-13, Core-14,Core-16, Core-17, Core-18, Core-19, Core-20, Core-21, and Core-22 inFIG. 9A-9B and FIG. 11A-11N. As another example, in some cases, the Tcell epitope polypeptide comprises the T-cell epitopes designated NS3-1,NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 9A-9B and FIG. 11A-11N. As another example, in somecases, the T cell epitope polypeptide comprises the T-cell epitopesdesignated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6, NS2-7, NS2-8,NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 9A-9B and FIG. 11A-11N. As another example,in some cases, the T cell epitope polypeptide comprises the T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1,NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, andNS4b-10 in FIG. 9A-9B and FIG. 11A-11N. As another example, in somecases, the T cell epitope polypeptide comprises the T-cell epitopesdesignated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8,NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2,NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1,NS5a-2, NS5b-1, and NS5b-2 in FIG. 9A-9B and FIG. 11A-11N. In somecases, the T-cell epitopes are contiguous. In some cases, any two T-cellepitopes are separated by linkers (e.g., a linker having a length offrom 1 amino acid to about 50 amino acids, e.g., from 1 amino acid to 5amino acids (aa), from 5 aa to 10 aa, from 10 aa to 15 aa, from 15 aa to20 aa, from 20 aa to 25 aa, from 25 aa to 30 aa, from 30 aa to 40 aa, orfrom 40 aa to 50 aa).

In some cases, the T cell epitope polypeptide comprises at least one HCVCD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope, whereepitopes are conserved among HCV genotypes 1 and 2. In some cases, the Tcell epitope polypeptide comprises at least one HCV CD4⁺ T cell epitopeand at least one HCV CD8⁺ T cell epitope, where epitopes are conservedamong HCV genotypes 1 and 3. In some cases, the T cell epitopepolypeptide comprises at least one HCV CD4⁺ T cell epitope and at leastone HCV CD8⁺ T cell epitope, where epitopes are conserved among HCVgenotypes 1, 2, and 3. In some cases, the T cell epitope polypeptidecomprises at least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺T cell epitope, where epitopes are conserved among HCV genotypes 1, 2,3, and 7. In some cases, the T cell epitope polypeptide comprises atleast one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope, where epitopes are conserved among HCV genotypes 1-7.

The T cell epitope polypeptide can have a length of from about 10 aminoacids to about 2000 amino acids; e.g., the T cell epitope polypeptidecan have a length of from 10 amino acids (aa) to 15 aa, from 15 aa to 20aa, from 20 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to 75 aa, from75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aato 400 aa, from 450 aa to 500 aa, from 500 aa to 550 aa, from 550 aa to600 aa, from 600 aa to 650 aa, from 650 aa to 700 aa, from 700 aa to 750aa, or from 750 aa to 800 aa. The T-cell epitope polypeptide can have alength of from about 25 amino acids to about 2000 amino acids, e.g.,from about 25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from 75 aato 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aa to250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to 400aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to 700 aa,from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to 1000 aa,from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aa to 1300aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500 aa to1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from 1800 aato 1900 aa, or from 1900 aa to 2000 aa. The T-cell epitope polypeptidecan have a length of from about 25 amino acids to about 3000 aminoacids, e.g., from about 25 amino acids (aa) to 50 aa, from 50 aa to 75aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa,from 200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa,from 350 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa,from 600 aa to 700 aa, from 700 aa to 800 aa, from 800 aa to 900 aa,from 900 aa to 1000 aa, from 1000 aa to 1100 aa, from 1100 aa to 1200aa, from 1200 aa to 1300 aa, from 1300 aa to 1400 aa, from 1400 aa to1500 aa, from 1500 aa to 1600 aa, from 1600 aa to 1700 aa, from 1700 aato 1800 aa, from 1800 aa to 1900 aa, from 1900 aa to 2000 aa, from 2000aa to 2250 aa, from 2250 aa to 2500 aa, from 2500 aa to 2750 aa, or from2750 aa to 3000 aa.

The T cell epitope polypeptide can have a length of from about 25 aminoacids to about 800 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The T-cell epitope polypeptide can have a length of from about 25 aminoacids to about 400 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, or from 350 aa to 400 aa. The T-cell epitopepolypeptide can have a length of 25 amino acids (aa), 26 aa, 27 aa, 28aa, 29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa, 36 aa, 37 aa, 38aa, 39 aa, 40 aa, 41 aa, 42 aa, 43 aa, 44 aa, 45 aa, 46 aa, 47 aa, 48aa, 49 aa, or 50 aa. The T cell epitope polypeptide can have a length offrom about 100 amino acids (aa) to 800 aa, e.g., from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The T cell epitope polypeptide can have a length of from 25 aa to 30 aa.The T cell epitope polypeptide can have a length of from 30 aa to 40 aa.The T cell epitope polypeptide can have a length of from 40 aa to 50 aa.The T-cell epitope polypeptide can have a length of from 50 aa to 60 aa(e.g., 50 aa, 51 aa, 52, aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa,59 aa, or 60 aa). The T cell epitope polypeptide can have a length offrom 60 aa to 70 aa. The T cell epitope polypeptide can have a length offrom 65 aa to 75 aa (e.g., 65, 66, 67, 68, 69, 70, 71, 72, 7, 74, or 75aa). The T cell epitope polypeptide can have a length of 70 aa. The Tcell epitope polypeptide can have a length of from 70 aa to 80 aa. The Tcell epitope polypeptide can have a length of from 80 aa to 90 aa. The Tcell epitope polypeptide can have a length of from 90 aa to 100 aa. TheT cell epitope polypeptide can have a length of from 100 aa to 105 aa(e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 aa). TheT cell epitope polypeptide can have a length of 100 aa. The T cellepitope polypeptide can have a length of from 10 amino acids (aa) to 50aa; e.g., from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to 25 aa,from 25 aa to 30 aa, from 30 aa to 35 aa, from 35 aa to 40 aa, from 40aa to 45 aa, or from 45 aa to 50 aa. The T cell epitope polypeptide canhave a length of from 10 amino acids (aa) to 20 aa, e.g., 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 aa.

HCV NS3 T-Cell Epitopes

In some cases, the T cell epitope polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS3 polypeptide. Examples of T-cell epitopespresent in NS3 polypeptides are depicted in FIG. 11A-11N, FIG. 9B, andFIG. 10A-10B.

The T cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:85).AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:85) is referred to in FIG. 10Aas “TP29.” In some cases, the T cell epitope polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:85); and has a length of from25 aa to 35 aa (e.g., 25 aa, 26 aa, 27 aa, 28 aa, 29 aa, 30 aa, 31 aa,32 aa, 33 aa, 34 aa, or 35 aa). In some cases, the T cell epitopepolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ IDNO:85); and has a length of 29 amino acids. Such a polypeptide caninclude NS3 T-cell epitopes designated NS3-3, NS3-4, and NS3-11 in FIG.9B and FIG. 11A-11N.

The T cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:87).AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:87) isreferred to in FIG. 10A as “TP52.” In some cases, the T cell epitopepolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:87); andhas a length of from 45 amino acids to 60 amino acids (e.g., 45 aa, 46aa, 47 aa, 48 aa, 49 aa, 50 aa, 51 aa, 52 aa, 53 aa, 54 aa, 55 aa, 56aa, 57 aa, 58 aa, 59 aa, or 60 aa). In some cases, the T cell epitopepolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:87); andhas a length of 52 amino acids. Such a polypeptide can include NS3T-cell epitopes designated NS3-3, NS3-4, NS3-5, and NS3-11 in FIG. 9Band FIG. 11A-11N.

The T cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:88); and has a length of from 65 amino acids to 80 aminoacids (e.g., 65 aa, 66 aa, 67 aa, 68 aa, 69 aa, 70 aa, 71 aa, 72 aa, 73aa, 74 aa, 75 aa, 76 aa, 77 aa, 78 aa, 79 aa, or 80 aa).KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:88) is referred to in FIG. 10A as “TP70.”

In some cases, the T cell epitope polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:88); and has a length of 70 amino acids. Such a polypeptidecan include NS3 T-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6,NS3-7, NS3-11, NS3-12, and NS3-13 in FIG. 9B and FIG. 11A-11N.

The T cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:89); and has alength of from 95 amino acids (aa) to 105 aa (e.g., 95 aa, 96 aa, 97 aa,98 aa, 99 aa, 100 aa, 101 aa, 102 aa, 103 aa, 104 aa, or 105 aa).VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:89) is referred toin FIG. 10A as “TP100.”

In some cases, the T cell epitope polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:89); and has alength of 100 amino acids. Such a polypeptide can include NS3 T-cellepitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 9B and FIG. 11A-11N.

The T cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:90);and has a length of from 171 amino acids (aa) to 180 aa (e.g., 171 aa,172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa, 179 aa, or 180aa. MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:90)is referred to in FIG. 10A as “TP171.”

In some cases, the T cell epitope polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:90);and has a length of 171 amino acids.

The T cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:129); and has a length of from 190 aminoacids (aa) to 200 aa (e.g., 190 aa, 191 aa, 192 aa, 193 aa, 194 aa, 195aa, 196 aa, 197 aa, 198 aa, 199 aa, or 200 aa.

In some cases, the T cell epitope polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:129); and has a length of 191 amino acids.

The T cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:91); and hasa length of from 215 amino acids (aa) to 235 aa (e.g., 215 aa, 216 aa,217 11, 218 aa, 219 aa, 220 aa, 221 aa, 222 aa, 223 aa, 224 aa, 225 aa,226 aa, 227 aa, 228 aa, 229 aa, 230 aa, 231 aa, 232 aa, 233 aa, 234 aa,or 235 aa). LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:91) isreferred to in FIG. 10A as “TP228.”

In some cases, the T cell epitope polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:91); and hasa length of 228 amino acids. Such a polypeptide can include NS3 T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 9B and FIG. 11A-11N.

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1265-1279 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1309-1323 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1401-1415 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1402-1412 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from11 amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa,15 aa, or 16 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1429-1439 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from11 amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa,15 aa, or 16 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1450-1464 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1453-1467 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1577-1591 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1306-1314 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1387-1394 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 1amino acids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12aa, 13 aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1405-1413 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1450-1458 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1457-1465 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVNS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1610-1618 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

HCV NS2 T-Cell Epitopes

In some cases, the T cell epitope polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS2 polypeptide. Examples of T-cell epitopespresent in NS2 polypeptides are depicted in FIG. 11A-11N, and FIG. 9A.

For example, the T cell epitope polypeptide can comprise an NS2 T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids955-974 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the T cell epitope polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 975-994 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the T cell epitope polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 985-1004 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the T cell epitope polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1015-1034 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the T cell epitope polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1035-1054 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the T cell epitope polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 924-933 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the T cell epitope polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 961-970 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the T cell epitope polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 989-997 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the T cell epitope polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 50 aa (e.g., from 10 aa to 25 aa, or from 25 aa to 50 aa) ofamino acids 955-1004 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa, orfrom 25 aa to 50 aa. In some cases, the T-cell epitope polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 955-1004of the amino acid sequence designated “Consensus” in FIG. 12A-12L, or acorresponding HCV NS2 amino acid sequence of any HCV genotype; and has alength of about 50 amino acids.

In some cases, the T cell epitope polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 553 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from 300 aato 400 aa, from 400 aa to 500 aa, or from 500 aa to 553 aa) of aminoacids 917-1469 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 and NS3 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa,from 25 aa to 50 aa, from 50 aa to 100 aa, from 100 aa to 200 aa, from200 aa to 300 aa, from 300 aa to 400 aa, from 400 aa to 500 aa, or from500 aa to 553 aa. In some cases, the T cell epitope polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 917-1469of the amino acid sequence designated “Consensus” in FIG. 12A-12L, or acorresponding HCV NS2 and NS3 amino acid sequence of any HCV genotype;and has a length of about 553 amino acids.

The T cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 0%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:86).LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:86) isreferred to in FIG. 10A as “TP50.” In some cases, the T cell epitopepolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:86); andhas a length of from 50 amino acids to 60 amino acids (e.g., 50 aa, 51aa, 52 aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60 aa).In some cases, the T cell epitope polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:86); andhas a length of 50 amino acids. Such a polypeptide can include NS2T-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 in FIG.9A and FIG. 11A-11N.

HCV NS4A T-Cell Epitopes

In some cases, the T cell epitope polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS4A polypeptide. Examples of T-cell epitopespresent in NS4A polypeptides are depicted in FIG. 11A-11N and FIG. 9B.

The T cell epitope polypeptide can comprise an NS4A T cell epitopecomprising an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids1683-1692 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS4A amino acid sequence of any HCVgenotype; and the NS4A T-cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

HCV NS4B T-Cell Epitopes

In some cases, the T cell epitope polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS4B polypeptide. Examples of T-cell epitopespresent in NS4B polypeptides are depicted in FIG. 11A-11N and FIG. 9B.

As one example, the T cell epitope polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1790-1801 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 12 aminoacids (aa) to 20 amino acids (e.g., 12 aa, 13 aa, 14 aa, 15 aa, 16 aa,17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the T cell epitope polypeptide can comprise an NS4BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1792-1802 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 11 aminoacids (aa) to 20 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15 aa,16 aa, 17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the T cell epitope polypeptide can comprise an NS4BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1898-1905 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 8 aminoacids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an NS4BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1921-1935 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the T cell epitope polypeptide can comprise an NS4BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1922-1941 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the T cell epitope polypeptide can comprise an NS4BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1928-1947 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the T cell epitope polypeptide can comprise an NS4BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1868-1876 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an NS4BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1927-1942 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 16 aminoacids (aa) to 20 amino acids (e.g., 16 aa, 17 aa, 18 aa, 19 aa, or 20aa).

As another example, the T cell epitope polypeptide can comprise an NS4BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1932-1940 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an NS4BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1948-1962 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

HCV NS5A T-Cell Epitopes

In some cases, the T cell epitope polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS5A polypeptide. Examples of T-cell epitopespresent in NS5A polypeptides are depicted in FIG. 11A-11N and FIG. 9B.

As one example, the T cell epitope polypeptide can comprise an NS5A Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2218-2232 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS5A amino acid sequence of any HCVgenotype; and the NS5A T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the T cell epitope polypeptide can comprise an NS5AT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2309-2317 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS5A amino acid sequence of any HCVgenotype; and the NS5A T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV NS5B T-Cell Epitopes

In some cases, the T cell epitope polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS5B polypeptide. Examples of T-cell epitopespresent in NS5B polypeptides are depicted in FIG. 11A-11N and FIG. 9B.

As one example, the T cell epitope polypeptide can comprise an NS5B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2847-2851 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS5B amino acid sequence of any HCVgenotype; and the NS5B T-cell epitope can have a length of from 5 aminoacids (aa) to 10 amino acids (e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10aa).

As another example, the T cell epitope polypeptide can comprise an NS5BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2602-2610 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS5B amino acid sequence of any HCVgenotype; and the NS5B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV Core T-Cell Epitopes

In some cases, the T cell epitope polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV core polypeptide. Examples of T-cell epitopespresent in HCV Core polypeptides are depicted in FIG. 11A-11N and FIG.9A.

As one example, the T cell epitope polypeptide can comprise an HCV coreT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1-20 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 11-30 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 21-40 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 39-63 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 23amino acids (aa) to 28 amino acids (e.g., 23 aa, 24 aa, 25 aa, 26 aa, 27aa, or 28 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 47-70 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 24amino acids (aa) to 29 amino acids (e.g., 24 aa, 25 aa, 26 aa, 27 aa, 28aa, or 29 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 61-80 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 71-90 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 81-100 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 91-110 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 101-115 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 111-130 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 125-139 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-150 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 151-170 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 161-180 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 35-44 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 43-51 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 51-59 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 129-137 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-140 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 150-158 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 154-162 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 168-176 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 177-187 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the T cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 178-187 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the T cell epitope polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 191 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, or from 150 aato 191 aa) of amino acids 1-191 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and has a length of from 10 amino acids(aa) to 25 aa, from 25 aa to 50 aa, from 50 aa to 100 aa, or from 100 aato 150 aa, or from 150 aa to 191 aa. In some cases, the T cell epitopepolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to amino acids1-191 of the amino acid sequence designated “Consensus” in FIG. 12A-12L,or a corresponding HCV core amino acid sequence of any HCV genotype; andhas a length of about 191 amino acids.

The T cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:90);and has a length of from 171 amino acids (aa) to 180 aa (e.g., 171 aa,172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa, 179 aa, or 180aa. In some cases, the T cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:90);and has a length of 171 amino acids. Such a polypeptide can include coreT-cell epitopes designated Core-1, Core-2, Core-3, Core-4, Core-5,Core-6, Core-7, Core-8, Core-9, Core-10, Core-11, Core-12, Core-13,Core-14, Core-16, Core-17, Core-18, Core-19, Core-20, Core-21, Core-22in FIG. 9A and FIG. 11A-11N.

HCV p7 T-Cell Epitopes

In some cases, the T cell epitope polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV p7 polypeptide. Examples of T-cell epitopespresent in HCV p7 polypeptides are depicted in FIG. 11A-11N or FIG. 9A.

As another example, the T cell epitope polypeptide can comprise an HCVp7 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 803-811 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV p7 amino acid sequence of any HCVgenotype; and the HCV p7 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

T-Cell Epitope Polypeptides Including HCV T-Cell Epitopes from More thanOne HCV Polypeptide Other than E1 and E2

As noted above, a T-cell epitope polypeptide can include T-cell epitopesfrom more than one HCV polypeptide other than E1 and E2.

As one example, a T cell epitope polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:92); and has a length of from 550 aminoacids (aa) to 560 aa (e.g., 550 aa, 551 aa, 552 aa, 553 aa, 554 aa, 555aa, 556 aa, 557 aa, 558 aa, 559 aa, or 560 aa).

QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:92) is referred to in FIG. 10A-10B as“TP553.”

In some cases, the T cell epitope polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:92); and has a length of 553 aminoacids. Such a polypeptide can include T-cell epitopes designated NS2-1,NS2-2, NS2-3, NS2-4, NS2-5, NS2-6, NS2-7, NS2-8, NS3-1, NS3-2, NS3-3,NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 inFIG. 9A-9B and FIG. 11A-11N. This polypeptide is also referred to as“TP553” (FIG. 12A-12D). In order to prevent self cleavage of the TP553polypeptide (amino acids 917-1469) (FIG. 11E-11G) at the NS2-NS3junction that is mediated by the catalytic domain of the NS2 protease(amino acids 917-1040), the histidine at position 966 (H966), a criticalresidue for NS2 protease activity, is mutated to alanine (H966A) (FIG.11E). See, e.g., Grakoui, A. et al. A second hepatitis C virus-encodedproteinase. Proc. Natl Acad. Sci. USA 90, 10583-10587 (1993); Hijikata,M. et al. Two distinct proteinase activities required for the processingof a putative nonstructural precursor protein of hepatitis C virus. J.Virol. 67, 4665-4675 (1993); and Lorenz. I C. Structure of the catalyticdomain of the hepatitis C virus NS2-3 protease. Nature. August 17;442(7104):831-5 (2006).

As another example, the T cell epitope polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aato 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa) the followingamino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:93); and has a length of from 778 amino acids (aa) to 790 aa(e.g., 778 aa, 779 aa, 780 aa, 781 aa, 782 aa, 783 aa, 784 aa, 785 aa,786 aa, 787 aa, 788 aa, or 790 aa).LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPG (SEQ ID NO:93) is referred to in FIG. 10B as “TP778.”

In some cases, the T cell epitope polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 25 amino acids(aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa, from 75aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aato 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aa to 550aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to 700 aa,from 700 aa to 750 aa, or from 750 aa to 778 aa) of the following aminoacid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:93); and has a length of from 25 amino acids (aa) to 50 aa,from 50 aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from300 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600aa to 700 aa, or from 700 aa to 778 aa. In some cases, the T-cellepitope polypeptide comprises an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity tothe following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:93); and has a length of 778 amino acids. Such a polypeptidecan include T-cell epitopes designated NS3-1, NS3-2, NS3-3, NS3-4,NS3-5, NS3-6, NS3-7, NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13,NS2-14, NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7,NS4b-8, NS4b-9, and NS4b-10 in FIG. 9B and FIG. 11A-11N.

As another example, the T cell epitope polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 1985 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 500 aa, from 500 aa to 750 aa, from 750aa to 1000 aa, from 1000 aa to 1500 aa, or from 1500 aa to 1985 aa) ofthe following amino acid sequence:APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDDSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGMTTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPPRKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGNNLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRFIRARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR (SEQ ID NO:94); this polypeptide isalso referred to as “TP1985” and is depicted in FIG. 10C.

In some cases, the T cell epitope polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVtSTWVLVGGVLAALAAYCISTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGMTTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPPRKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGNNLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR (SEQ ID NO:94); and has a length of1985 amino acids. Such a polypeptide can include T-cell epitopesdesignated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8,NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2,NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1,NS5a-2, NS5b-1, NS5b-2 in FIG. 9A-9B and FIG. 11A-11N.

Additional T-Cell Epitopes

As discussed above, an immunogenic composition of the present disclosureincludes: a) an HCV E1/E2 heterodimer; b) a T cell epitope polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide); and c) a CDN. The one or more T-cell epitopescan include one or more T-cell epitopes present in: a) an HCV NS3polypeptide; b) an HCV NS2 polypeptide; c) an HCV NS4A polypeptide; d)an HCV NS4B polypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5Bpolypeptide; g) an HCV core polypeptide; or h) an HCV p7 polypeptide. Insome cases, the one or more T-cell epitopes are T-cell epitopes presentin an HCV NS3 polypeptide. In some cases, the T cell epitope polypeptidefurther comprises one or more T cell epitopes present in: a) choleratoxin or toxoid; and/or b) tetanus toxin or toxoid; and/or c) diphtheriatoxin or toxoid; and/or d) a meningococcal outer membrane protein.

Thus, in some cases, an immunogenic composition of the presentdisclosure includes: a) an HCV E1/E2 heterodimer; b) a T-cell epitopepolypeptide that comprises one or more T-cell epitopes, where the one ormore T-cell epitopes are T-cell epitopes present in: i) one or more ofan HCV NS3 polypeptide, an HCV NS2 polypeptide, an HCV NS4A polypeptide,an HCV NS4B polypeptide, an HCV NS5A polypeptide, an HCV NS5Bpolypeptide, an HCV core polypeptide, and an HCV p7 polypeptide; and ii)one or more of cholera toxin or toxoid, tetanus toxin or toxoid,diphtheria toxin or toxoid, and a meningococcal outer membrane protein;and c) a CND.

A T helper tetanus toxin epitope or other bacterial T-cell epitope couldbe fused (e.g., by recombinant expression) or chemically conjugated tothe T cell epitope polypeptide, or can be unconjugated (e.g., providedas a separate polypeptide), to further enhance both T and B cellresponses to both the T-cell epitopes present in the T cell epitopepolypeptide and in the E1/E2 polypeptides. Alternatively, the whole orpart of the detoxified toxin (“toxoid”) can be used, wherein specificamino acids of the toxins are mutated to render the toxins inactive,thereby generating toxoids. Methods of generating toxoids are well knownin the art. Examples of bacterial epitopes include the use of diphtheriatoxoid, meningococcal outer membrane protein, or mutant diphtheriaprotein CRM197 (see, e.g.: world-wide-website:Medscape.com/viewarticle/431127).

In some cases, a suitable tetanus toxoid polypeptide comprises the aminoacid sequence QYIKANSKFIGIFE (SEQ ID NO:130). In some cases, a suitabletetanus toxoid polypeptide comprises the amino acid sequenceQYIKANSKFIGITE (SEQ ID NO:131).

In some cases, a T cell epitope polypeptide can comprise cholera toxin(or toxoid) epitope. In some cases, a suitable T-cell epitopepolypeptide comprising a cholera toxoid epitope comprises a fragment ofcholera toxin-B subunit (CT-B), e.g., a fragment of from 5 amino acidsto 25 amino acids, or from 25 amino acids to 50 amino acids, of thefollowing amino acid sequence: MIKLKFGVFF TVLLSSAYAH GTPQNITDLCAEYHNTQIHT LNDKIFSYTE SLAGKREMAI ITFKNGATFQ VEVPGSQHID SQKKAIERMKDTLRIAYLTE AKVEKLCVWN NKTPHAIAAI SMAN (SEQ ID NO:132).

In some cases, a T cell epitope polypeptide can comprise a tetanus toxin(or toxoid) T-cell epitope. In some cases, a suitable T cell epitopepolypeptide comprising a tetanus toxin T-cell epitope comprises theamino acid sequence: ILMQYIKANSKFIGI (SEQ ID NO:133); and has a lengthof from 15 amino acids to 20 amino acids. In some cases, a suitable Tcell epitope polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: VNNESSE (SEQ ID NO:134). In somecases, a suitable T cell epitope polypeptide comprising a tetanus toxinT-cell epitope comprises the amino acid sequence: PGINGKAIHLVNNESSE (SEQID NO:135). In some cases, a suitable T cell epitope polypeptidecomprising a tetanus toxin T-cell epitope comprises the amino acidsequence: PNRDIL (SEQ ID NO:136). In some cases, a suitable T cellepitope polypeptide comprising a tetanus toxin T-cell epitope comprisesthe amino acid sequence: FIGITEL (SEQ ID NO:137). In some cases, asuitable tetanus toxin T-cell epitope comprises the amino acid sequence:SYFPSV (SEQ ID NO:138). In some cases, a suitable T cell epitopepolypeptide comprising a tetanus toxin T-cell epitope comprises theamino acid sequence: NSVDDALINSTKIYSYFPSV (SEQ ID NO:139). In somecases, a suitable T cell epitope polypeptide comprising a tetanus toxinT-cell epitope comprises the amino acid sequence: IDKISDVSTIVPYIGPALNI(SEQ ID NO:140).

In some cases, a T cell epitope polypeptide can comprise a diphtheriatoxin T-cell epitope In some cases, a suitable T-cell epitopepolypeptide comprising a diphtheria toxin T-cell epitope comprises theamino acid sequence: QSIALSSLMVAQAIP (SEQ ID NO:141); and has a lengthof from 15 amino acids to 20 amino acids. In some cases, a suitable Tcell epitope polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: PVFAGANYAAWAVNVAQVI (SEQ ID NO:142).In some cases, a suitable T cell epitope polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:VHHNTEEIVAQSIALSSLMV (SEQ ID NO:143). In some cases, a suitable T cellepitope polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: QSIALSSLMVAQAIPLVGEL (SEQ ID NO:144).In some cases, a suitable T cell epitope polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:VDIGFAAYNFVESIINLFQV (SEQ ID NO:145). In some cases, a suitable T cellepitope polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: QGESGHDIKITAENTPLPIA (SEQ ID NO:146).In some cases, a suitable T cell epitope polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:GVLLPTIPGKLDVNKSKTHI (SEQ ID NO:147). In some cases, a suitable T cellepitope polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence of CRM197 (see, e.g., Giannini et al.(1984) Nucl. Acids. Res. 12:4063).

The amino acid sequence of CRM197 is as follows:

(SEQ ID NO: 148) laddvvdssksfvmenfssyhgtkpgyvdsiqkgiqkpksgtqgnydddwkefystdnkydaagysydnenplsgkaggvvkvtypgltkvlalkvdnaetikkelglslteplmeqvgteefikrfgdgasrvvlslpfaegsssveyinnweqakalsveleinfetrgkrgqdamyeymaqacagnrvrrsygsslscinldwdvirdktktkieslkehgpiknkmsespnktvseekakqyleefhqtalehpelselktvtgtnpvfaganyaawavnvaqvidsetadnlekttaalsilpgigsvmgiadgavhhnteeivaqsialsslmvaqaiplygelvdigfanynfvesiinlfqvvhnsynrpayspghktqpflhdgyayswntvedsiirtgfqgesghdikitaentplpiagvllptipgkldvnkskthisvngrkirmrcraidgdvtfcrpkspvyvgngvhanlhvafhrsssekihsneissdsigvlgyqktvdhtkvnsklslffeiks.

In some cases, a T cell epitope polypeptide can comprise a tetanus toxinT-cell epitope and a diphtheria toxin T-cell epitope. In some of thesecases, the T cell epitope polypeptide can comprise the amino acidsequence: IMQYIKANSKFIGIQSIALSSLMVAQ (SEQ ID NO:149); and can have alength of from 26 amino acids to 30 amino acids.

Mixtures of T-Cell Epitope Polypeptides (T-Cell Epitope Polypeptides)

In some cases, an immunogenic composition of the present disclosurecomprises two or more different T-cell epitope polypeptides comprising aT-cell epitope present in an HCV protein other than E1 and E2 (e.g., amixture of two or more different T-cell epitope polypeptides comprisinga T-cell epitope present in an HCV protein other than E1 and E2).

For example, in some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimeric polypeptide; b) twoor more different T-cell epitope polypeptides comprising a T-cellepitope present in an HCV protein other than E1 and E2; and c) a CDN. Insome cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E2 polypeptide; b) two or more different T-cellepitope polypeptides comprising a T-cell epitope present in an HCVprotein other than E1 and E2; and c) a CDNs.

For example, the two or more different T cell epitope polypeptides caninclude: i) a first T cell epitope polypeptide comprising an amino acidsequence having at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, or 100%, amino acid sequence identity to TP29, and having alength of from 29 amino acids to 35 amino acids; and ii) a second T cellepitope polypeptide comprising an amino acid sequence having at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%,amino acid sequence identity to TP50, and having a length of from 50amino acids to 55 amino acids.

As another example, the two or more different T cell epitopepolypeptides can include: i) a first T cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second T cell epitope polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP52, andhaving a length of from 52 amino acids to 60 amino acids.

As another example, the two or more different T cell epitopepolypeptides can include: i) a first T cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a T cell epitope polypeptide comprising an amino acidsequence having at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, or 100%, amino acid sequence identity to TP70, and having alength of from 70 amino acids to 75 amino acids.

As another example, the two or more different T cell epitopepolypeptides can include: i) a first T cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second T cell epitope polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP100, andhaving a length of from 100 amino acids to 110 amino acids.

As another example, the two or more different T cell epitopepolypeptides can include: i) a first T cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second T cell epitope polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP171, andhaving a length of from 171 amino acids to 180 amino acids.

As another example, the two or more different T cell epitopepolypeptides can include: i) a first T cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a T cell epitope polypeptide comprising an amino acidsequence having at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, or 100%, amino acid sequence identity to TP228, and having alength of from 228 amino acids to 235 amino acids.

As another example, the two or more different T cell epitopepolypeptides can include: i) a first T cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second T cell epitope polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP553, andhaving a length of from 553 amino acids to 565 amino acids.

As another example, the two or more different T cell epitopepolypeptides can include: i) a first T cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second T cell epitope polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP778, andhaving a length of from 778 amino acids to 785 amino acids.

Pharmaceutically Acceptable Excipients

An immunogenic composition of the present disclosure can include apharmaceutically acceptable excipient. A wide variety ofpharmaceutically acceptable excipients is known in the art and need notbe discussed in detail herein. Pharmaceutically acceptable excipientshave been amply described in a variety of publications, including, forexample, A. Gennaro (2000) “Remington: The Science and Practice ofPharmacy”, 20th edition, Lippincott, Williams, & Wilkins; PharmaceuticalDosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds7th ed., Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. PharmaceuticalAssoc.

In some cases, a pharmaceutically acceptable excipient is an aqueousbuffer. Thus, an immunogenic composition of the present disclosure caninclude an aqueous buffer. Suitable aqueous buffers include, but are notlimited to, acetate, succinate, citrate, and phosphate buffers varyingin strengths from about 5 mM to about 100 mM. In some cases, the aqueousbuffer includes reagents that provide for an isotonic solution. Suchreagents include, but are not limited to, sodium chloride; and sugarse.g., mannitol, dextrose, sucrose, and the like. In some embodiments,the aqueous buffer further includes a non-ionic surfactant such aspolysorbate 20 (TWEEN®20) or polysorbate 80 (TWEEN®80). For example, animmunogenic composition of the present disclosure in an aqueous buffercan include, e.g., from about 0.01% to about 0.05% polysorbate-20(TWEEN®20) non-ionic detergent. Optionally the formulations may furtherinclude a preservative. Suitable preservatives include, but are notlimited to, a benzyl alcohol, phenol, chlorobutanol, benzalkoniumchloride, and the like. In many cases, the formulation is stored atabout 4° C. Formulations may also be lyophilized, in which case theygenerally include cryoprotectants such as sucrose, trehalose, lactose,maltose, mannitol, and the like. Lyophilized formulations can be storedover extended periods of time, even at ambient temperatures. In somecases, the aqueous buffer further includes a non-ionic surfactant. Insome cases, the aqueous buffer includes the non-ionic surfactant Triton™X-100, e.g., 0.1% Triton™ X-100.

Cyclic Dinucleotides

In some cases, a CDN suitable for use in an immunogenic composition ofthe present disclosure is of Formula (I):

wherein:

A is S or O;

X is S, N, O, CH₂;

Y, Y′ is NH, CH₂, O;

Z, Z′ is NH, CH₂, O;

R1 represents hydrogen or NH₂ which may be substituted;

R2 is hydrogen or absent;

R3 represents NH₂, O, OH, H, or a halogen;

R4 represents hydrogen, halogen, or a straight or branched C₁-C₆ alkylgroup which may optionally be substituted;

R5 represents hydrogen, OH or a straight or branched C₁-C₆ alkyl chainor C₁-C₆ straight or branched alkoxy chain which may optionally besubstituted;

is a single or double bond;

or conjugates thereof, and salts or solvates thereof. See, e.g., US2008/0286296.

In formula (I), the purine residue is in some cases a guanine (G),adenine (A), xanthine or hypoxanthine (X), or inosine (I) residue. Thecompound can have identical purine residues, e.g. c-diGMP, c-diAMP,c-dilMP, or c-dXMP, or can contain different purine residues, e.g.c-GpAp, c-Gplp, c-GpXp, c-Aplp, c-ApXp, or c-lpXp. Further, R5 is insome cases an OH group. In addition, X is in some cases an oxygen atom.In one embodiment, Y, Y′, Z, and Z′ are an oxygen atom, O. Thus, in oneembodiment, the compound of formula (I) is a cyclic bis(3′-5′)diguanylicacid (c-diGMP) or conjugates thereof or a cyclic bis(3′-5′)diadenylicacid (c-diAMP) or conjugates thereof, or salts or solvates thereof. Inone embodiment, the compound of formula (I) is cyclic Bis(3′-5′)adenylicacid, which is also referred to as c-di-AMP; or the pegylated conjugate.With the term “which may be substituted” is meant the substitution witha straight or branched C1-C6 alkyl group or a straight or branched C1-C6alkoxy group and/or with a halogen, hydroxyl group or carboxyl group.

In some cases, a CDN suitable for use in an immunogenic composition ofthe present disclosure is selected from the group consisting of cyclicdi-adenosine monophosphate (c-di-AMP), cyclic di-guanosine monophosphate(c-di-GMP), and cyclic guanosine monophosphate-adenosine monophosphate(cGAMP). In some cases, a CDN suitable for use in an immunogeniccomposition of the present disclosure is cGAMP (2′-3′-cyclic GMP-AMP) orcGAMP (3′-3′-cyclic GMP-AMP). In some cases, a CDN suitable for use inan immunogenic composition of the present disclosure is cGAMP(2′-3′-cyclic GMP-AMP). In some cases, a CDN suitable for use in animmunogenic composition of the present disclosure is cGAMP (3′-3′-cyclicGMP-AMP).

In some cases, a CDN suitable for use in an immunogenic composition ofthe present disclosure is of Formula (II):

where:

A, C, A′ and C′ are independently selected from NH, O, and S;

X, Y, X′, and Y′ are independently selected from O or S;

Z and Z′ are independently selected from O, S, NH, and CH₂; and

B₁ and B₂ are independently a purine selected from:

where:

Q is hydrogen or NH₂;

Nitrogen is optionally substituted with a C₁-C₆ alkyl or a C₁-C₆ acylgroup; and

R is O or S.

In some cases, a CDN suitable for inclusion in an immunogeniccomposition of the present disclosure is a fluorinated CND. In somecases, the fluorinated CDN is 2′-F-c-diGMP having the followingstructure:

Immunogenic Composition Comprising HCV E1/E2, HCV E2, or HCV E1 and anArchaeal Glycolipid

The present disclosure provides an immunogenic composition comprising:a) an HCV E1E2 heterodimer; and b) an archaeosome. The presentdisclosure provides an immunogenic composition comprising: a) an HCV E2polypeptide; and b) an archaeosome. The present disclosure provides animmunogenic composition comprising: a) an HCV E1 polypeptide; and b) anarchaeosome.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces an immuneresponse in the individual to one or more HCV genotypes. In some cases,an immunogenic composition of the present disclosure, when administeredto an individual in need thereof, induces an immune response in theindividual to one or more HCV genotypes, where the immune response isgreater than the immune response induced by administration of a controlcomposition comprising the HCV E1/E2 heterodimer (or E1 polypeptide, orE2 polypeptide) but lacking the archaeosome.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces CD8⁺ CTLsspecific for HCV, where the number of HCV-specific CD8⁺ CTLs induced isat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, higher than the number of HCV-specific CD8⁺ CTLs induced byadministration of a control composition (e.g., a composition comprisingthe HCV E1/E2 heterodimer but lacking the archaeosome; a compositioncomprising an E1 polypeptide but lacking the archaeosome; a compositioncomprising an E2 polypeptide but lacking the archaeosome).

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces CD4⁺ T cellsspecific for HCV, where the number of HCV-specific CD4⁺ T cells inducedis at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, higher than the number of HCV-specific CD4⁺ T cells induced byadministration of a control composition (e.g., a composition comprisingthe HCV E1/E2 heterodimer but lacking the archaeosome; a compositioncomprising an E1 polypeptide but lacking the archaeosome; a compositioncomprising an E2 polypeptide but lacking the archaeosome).

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces productionof HCV-specific CD4⁺ T cells and CD8⁺ T cells in the individual, wherethe number of HCV-specific CD4⁺ T cells and/or CD8⁺ T cells isincreased, such that the percent of total peripheral CD4⁺ and/or CD8⁺ Tcells that is HCV-specific is from 0.01% to 0.05%, from 0.05% to 0.10%,from 0.10% to 0.125%, from 0.125% to 0.25%, from 0.25% to from 0.50%, or0.5% to 10% (e.g., from 0.5% to 1%, from 1% to 2%, from 2% to 5%, orfrom 5% to 10%). The number of HCV-specific CD4⁺ T cells and CD8⁺ Tcells in a control individual (e.g., an individual not infected withHCV) not treated with the immunogenic composition would be undetectable.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, increases the numberof HCV E1/E2-specific CD4⁺ T cells and CD8⁺ T cells in the individual byat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, compared to the number of HCV E1/E2-specific CD4⁺ T cells andCD8⁺ T cells in the individual induced by administration of a controlcomposition comprising the HCV E1/E2 heterodimer (or HCV E2 polypeptide,or HCV E1 polypeptide) but lacking the archaeosome, or compared to thenumber of HCV E1/E2-specific CD4⁺ T cells and CD8⁺ T cells in theindividual before administration of the immunogenic composition.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces helper Tlymphocytes (e.g., CD4⁺ T cells) specific for HCV, where the number ofHCV-specific helper T lymphocytes induced is at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 7.5-fold, at least 10-fold, at least 20-fold, at least50-fold, or at least 100-fold, or more than 100-fold, higher than thenumber of HCV-specific helper T cells induced by administration of acontrol composition comprising the HCV E1/E2 heterodimer (or HCV E2polypeptide, or HCV E1 polypeptide) but lacking the archaeosome, orcompared to the number of HCV-specific CD4⁺ T cells in the individualbefore administration of the immunogenic composition.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces antibodyspecific for HCV, where the level of HCV-specific antibody induced is atleast at high as the level of HCV-specific antibody induced byadministration of a control composition comprising the HCV E1/E2heterodimer (or HCV E2 polypeptide, or HCV E1 polypeptide) but lackingthe archaeosome.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces antibodyspecific for HCV, where the level of HCV-specific antibody induced is atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, higher than the level of HCV-specific antibody induced byadministration of a control composition comprising the HCV E1/E2heterodimer (or HCV E2 polypeptide, or HCV E1 polypeptide) but lackingthe archaeosome, or compared the level of HCV-specific antibody in theindividual before administration of the immunogenic composition.

An immunogenic composition of the present disclosure, when administeredto an individual in need thereof, induces an immune response (e.g., acellular immune response) in the individual to one or more HCVgenotypes. In some cases, an immunogenic composition of the presentdisclosure, when administered to an individual in need thereof, inducesan immune response in the individual to HCV genotype 1. In some cases,an immunogenic composition of the present disclosure, when administeredto an individual in need thereof, induces an immune response in theindividual to HCV genotype 2. In some cases, an immunogenic compositionof the present disclosure when administered to an individual in needthereof, induces an immune response in the individual to HCV genotype 3.In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 1 and HCV genotype 3. In somecases, an immunogenic composition of the present disclosure, whenadministered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 1, HCV genotype 2, and HCVgenotype 3. In some cases, an immunogenic composition of the presentdisclosure, when administered to an individual in need thereof, inducesan immune response in the individual to HCV genotype 1, HCV genotype 2,HCV genotype 3, and HCV genotype 7. In some cases, an immunogeniccomposition of the present disclosure, when administered to anindividual in need thereof, induces an immune response in the individualto HCV genotype 1, HCV genotype 2, HCV genotype 3, HCB genotype 4, HCVgenotype 5, HCV genotype 6, and HCV genotype 7.

HCV E1E2 Heterodimers, HCV E2 Polypeptides, and HCV E1 Polypeptides.

The present disclosure provides an immunogenic composition comprising:a) an HCV E1E2 heterodimer; and b) an archaeosome. The presentdisclosure provides an immunogenic composition comprising: a) an HCV E2polypeptide; and b) an archaeosome. The present disclosure provides animmunogenic composition comprising: a) an HCV E1 polypeptide; and b) anarchaeosome. Suitable HCV E1E2 heterodimers, HCV E2 polypeptides, andHCV E1 polypeptides are as described above.

Archaeal Lipids and Archaeosomes

An archaeal lipid suitable for use in an immunogenic composition of thepresent disclosure comprises a polar lipid based on a 2,3-dialkylglycerol skeleton. These 2, 3-dialkylglycerol groups areisoprenoid and the simplest molecules are derivatives or2,3-dibiphytanyl-O-sn-glycerol (archeol); for instance, two isoprenoidunits of 20 carbons joined at positions sn-2 and sn-3 of glycerol. Thesealkyl chains are generally saturated; nevertheless, some forms havedouble bonds in different positions. These lipids have one or two groupsof polar head, which may be different with units 2, 3-sn-glycerol joinedby C40 alkyl components which are also isoprenoid molecules. Forinstance, calarcheol (so called because it is the predominant form insome thermophile archaebacteria), has two C40 isoprenoid units bondedfrom positions 2 to 3′ and from position 3 to 2′.

In some cases, an archaeal adjuvant suitable for use in an immunogeniccomposition of the present disclosure comprises multivalent cations inassociation with aggregates of a plurality of spherical archaeal polarlipid structures containing aqueous compartments (e.g., an “AMVADstructure”), where the archaeal polar lipid is a total polar lipidsextract or archaetidyl glycerophosphate-O-methyl, obtained from anarchaeal species. The multivalent cations can be divalent or trivalentcations. The multivalent cations can be divalent Ca²⁺ or Mg²⁺, ortrivalent Al³⁺. The Ca²⁺ can be provided as CaCl₂. The Al³⁺ can beprovided as AlCl₃ or AlK(SO₄)₂. In some cases, the total polar lipidsextract from an archaeal species is mixed with neutral lipids from thearchaeal species. See, e.g., U.S. Patent Publication No. 2013/0195932.

In some cases, lipids suitable for use in an immunogenic composition ofthe present disclosure comprises1,2-di-O-hexadecyl-sn-glycero-3-phosphatidylcholine and1,2-di-O-phytanyl-sn-glycero-3-phosphatidylethanolamine. In some cases,the 1,2-di-O-hexadecyl-sn-glycero-3-phosphatidylcholine and1,2-di-O-phytanyl-sn-glycero-3-phosphatidylethanolamine form uniformlysized particles; for example, the particles can comprise: liposomes,nanoliposomes, niosomes, microspheres, nanospheres, nanoparticles,micelles or archaeosomes.

In some cases, an archaeosome comprises at least one polar syntheticlipid, where the at least one polar synthetic lipid comprises at leastone carbohydrate or anionic group linked by covalent bonding to at leastone free hydroxyl group of an archaeal core lipid. In some cases, thearchaeal core lipid is archaeol (2,3-di-O-diphytanyl-sn-glycerol). Insome cases, the archaeal core lipid is caldarchaeol(2,2′,3,3′-tetra-O-dibiphytanyl-sn-diglycerol). In some cases, thecarbohydrate group is selected from the group consisting of:β-D-Glc-(1,6)-β-D-Glc-; β-D-Glc-(1,6)-α-D-Glc-; α-D-Glc-(1,6)-β-D-Glc-;β-D-Glc-(1,4)-β-D-Glc-; α-D-Glc-(1,4)-β-D-Glc-; β-D-Gal-(1,4)-β-D-Glc-;α-D-Gal-(1,6)-β-D-Glc-; β-D-Glc-(1,6)-β-D-Glc-(1,6)-β-D-Glc-;α-D-Glc-(1,4)-α-D-Glc-(1,4)-β-D-Glc-;α-D-Man-(1,2)-α-D-Man-(1,2)-α-D-Man-; andα-D-Man-(1,2)-α-D-Man-(1,2)-α-D-Man-(1,2)-α-D-Man-. In some cases, thecarbohydrate group comprises two or three β-D-Glc-units in (1,6)linkage. In some cases, the carbohydrate group is a Galactose-Glucose(gal-glc) group. In some cases, the anionic group is selected from thegroup consisting of phosphoserine, phosphoethanolamine, phosphoinositoland phosphoglycerol. In some cases, the at least one anionic lipid isselected from the group consisting of archaetidylglycerol,archaetidylglycerolphosphate-methyl, archaetidylserine, andarchaetidylinositol. In some cases, the archaeosome comprises at leastone conventional lipid. In some cases, the at least one conventionallipid is selected from a group consisting of phosphatidylglycerol,phosphatidylserine, sulfoquinovosyl diacylglycerol (SQDG), andcholesterol. In some cases, the at least one conventional lipidcomprises cholesterol, and wherein cholesterol is present in an amountof between 10 and 45 mol % of the total lipid composition. In somecases, the phosphatidylglycerol is present in an amount of between 20and 80 mol % of the lipid composition. In some cases, thephosphatidylserine is present in an amount of between 10 and 30 mol % ofthe lipid composition. In some cases, the at least one polar syntheticlipid comprises at least one synthetic immunoactive glycolipid and atleast one anionic lipid, and the archaeosome further comprises at leastone stabilizing lipid. In some cases, the at least one polar syntheticlipid comprises caldarchaeol having one carbohydrate head group and oneanionic head group. In some cases, the carbohydrate head group comprisesgentiobiose and the anionic head group comprises phosphoinositol. Insome cases, the at least one polar synthetic lipid comprises a firstcaldarchaeol having two carbohydrate head groups and a secondcaldarchaeol having two anionic head groups, and wherein the at leastone stabilizing lipid is the first and/or second caldarchaeol. In somecases, the at least one polar synthetic lipid comprisesgentiotriose-archaeol and wherein the at least one stabilizing lipidcomprises cholesterol and at least one of phosphatidylethanolamine,archaetidylglycerol, archaetidylserine orarchaetidylglycerolphosphate-methyl.

Caldarchaeol is also known as dibiphytanyldiglycerol tetraether. Twoglycerol units are linked together by two strains that consist of twophytanes linked together to form a linear chain of 32 carbon atoms.Caldarchaeol has the following structure:

Archaeal lipids can be obtained from any archaea of the phylaEuryarchaeota, Crenarchaeota, Korarchaeota, or, Nanoarchaea. Archaeallipids can be obtained from any archaea of the genus Thermococcus,Sulfolobus, Halobacterium, Methanococcus, Ferroglobus, Thermoplasma,Archaeoglobus, Haloquadratum, or Halorubrum. Suitable sources ofarchaeal lipids include, but are not limited to, Thermus aquaticus,Thermus thermophilus; Methanobrevibacter smithii; Thermoplasmaacidophilum; a Sulfolobus species, e.g. Sulfolobus acidocaldarius,Sulfolobus solfataricus, Sulfolobus islandicus, Sulfolobus tokodaii,etc.; a Pyrobaculum species, e.g. Pyrobaculum islandicum or Pyrobaculumaerophilum; a Methanococcus species, e.g., Methanocaldococcus vulcanius,Methanocaldococcus jannaschii, Methanococcus acolicus, Methanococcusvoltae; or a Halobacterium species such as Halobacterium salinarum;Methanopyrus kandleri; Methanobacterium espanolae; Methanosphaerastadtmanae; Methanosarcina mazei; Natronobacterium magadii; etc.

Total polar lipids (TPL) can be extracted from archaea and collected asthe acetone-insoluble fraction. Choquet et al. (1994) Appl. Microbiol.Biotechnol. 42:375; Bligh and Dyer (1959) Can. J. Biochem. Physiol.37:911. The polar lipids consist of regularly branched, and usuallyfully saturated, phytanyl chains of 20, 25, or 40 carbon length, withthe 20 and 40 being most common. Archaeosomes can be prepared byhydrating TPL in a buffer (e.g., phosphate-buffered saline). TheTPL-buffer solution can be sonicated (e.g., at 60 Hz for 10 min).

TPL can be extracted from archaea by stirring the cells (which may belyophilized) with chloroform-methanol (2:1, v/v) for 1 hour at roomtemperature. The suspension is passed through a sintered glass filter,and the residue reextracted for an additional hour. Combined filtratesare evaporated, taken up in chloroform-methanol-water (60:30:4.5,v/v/v), and passed through Sephadex G-25 for removal of nonlipidcontaminations. Langworthy et al. (1977) J. Bacteriol. 130:1326.

The mean diameter of archaeosomes in an archaeosomal formulation canrange from about 50 nm to 600 nm, e.g., from 50 nm to 100 nm, from 100nm to 150 nm, from 150 nm to 200 nm, from 200 nm to 250 nm, from 250 nmto 300 nm, from 300 nm to 400 nm, from 400 nm to 450 nm, from 450 nm to500 nm, from 500 nm to 550 nm, or from 550 nm to 600 nm.

T-Cell Epitope Polypeptides

In some cases, one or both of the polypeptide chains of the E1/E2heterodimer present in an immunogenic composition of the presentdisclosure can include a T-cell epitope polypeptide. In some cases, anE2 polypeptide present in an E1/E2 heterodimer present in an immunogeniccomposition of the present disclosure includes a T-cell epitopepolypeptide. In some cases, an E1 polypeptide present in an E1/E2heterodimer present in an immunogenic composition of the presentdisclosure includes a T-cell epitope polypeptide. In some cases, an HCVE2 polypeptide present in an immunogenic composition of the presentdisclosure includes a T-cell epitope polypeptide. In some cases, an HCVE1 polypeptide present in an immunogenic composition of the presentdisclosure includes a T-cell epitope polypeptide. In these embodiments,the T-cell epitope is covalently linked to the E1 and/or E2 polypeptide.For example, in some cases, the T-cell epitope is covalently linked tothe amino terminus (N-terminus) of the HCV E1 polypeptide. In somecases, the T-cell epitope is covalently linked to the carboxyl terminus(C-terminus) of the HCV E1 polypeptide. Thus, e.g., in some cases, animmunogenic composition of the present disclosure comprises: a) a fusionpolypeptide comprising, in order from N-terminus to C-terminus: i) anHCV E1 polypeptide; and ii) a T-cell epitope polypeptide; and b) anarchaeosome. In some cases, an immunogenic composition of the presentdisclosure comprises: a) a fusion polypeptide comprising, in order fromN-terminus to C-terminus: i) an HCV E2 polypeptide; and ii) a T-cellepitope polypeptide; and b) an archaeosome. In some cases, animmunogenic composition of the present disclosure comprises: a) a fusionpolypeptide comprising, in order from N-terminus to C-terminus: i) aT-cell epitope polypeptide; and ii) an HCV E1 polypeptide; and b) anarchaeosome. In some cases, an immunogenic composition of the presentdisclosure comprises: a) a fusion polypeptide comprising, in order fromN-terminus to C-terminus: i) a T-cell epitope polypeptide; and ii) anHCV E2 polypeptide; and b) an archaeosome. In some cases, an immunogeniccomposition of the present disclosure comprises: a) an HCV E1E2heterodimer comprising i) a fusion polypeptide comprising, in order fromN-terminus to C-terminus: an HCV E1 polypeptide; and a T-cell epitopepolypeptide; and ii) an HCV E2 polypeptide; and b) an archaeosome. Insome cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1E2 heterodimer comprising i) a fusion polypeptidecomprising, in order from N-terminus to C-terminus: an HCV E2polypeptide; and a T-cell epitope polypeptide; and ii) an HCV E2polypeptide; and b) an archaeosome. In some cases, an immunogeniccomposition of the present disclosure comprises: a) an HCV E1E2heterodimer comprising i) a fusion polypeptide comprising, in order fromN-terminus to C-terminus: a T-cell epitope polypeptide; and an HCV E1polypeptide; and ii) an HCV E2 polypeptide; and b) an archaeosome. Insome cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1E2 heterodimer comprising i) a fusion polypeptidecomprising, in order from N-terminus to C-terminus: a T-cell epitopepolypeptide; and an HCV E2 polypeptide; and ii) an HCV E1 polypeptide;and b) an archaeosome.

In some cases, an immunogenic composition of the present disclosurecomprises a T-cell epitope polypeptide, where the T-cell epitopepolypeptide is not covalently linked to the HCV E1/E2 heterodimer, theHCV E1 polypeptide or the HCV E2 polypeptide. For example, in somecases, an immunogenic composition of the present disclosure comprises:a) an HCV E1/E2 heterodimer; b) an archaeosome; and c) a T-cell epitopepolypeptide. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E2 polypeptide; b) an archaeosome; andc) a T-cell epitope polypeptide. In some cases, an immunogeniccomposition of the present disclosure comprises: a) an HCV E1polypeptide; b) an archaeosome; and c) a T-cell epitope polypeptide.

Suitable T-cell epitope polypeptides are as described above.

Compositions Comprising a T-Cell Epitope Polypeptide Comprising T-CellEpitopes Present in an HCV Polypeptide Other than E1 and E2

The present disclosure provides an immunogenic composition comprising:a) a T-cell epitope polypeptide comprising T-cell epitopes present in anHCV polypeptide other than E1 and E2; and b) a CDN. The presentdisclosure provides an immunogenic composition comprising: a) a T-cellepitope polypeptide comprising T-cell epitopes present in an HCVpolypeptide other than E1 and E2; and b) an archaeosome.

Suitable T-cell epitope polypeptides are as described above. SuitableCDNs are as described above. Suitable archaeosomes are as describedabove.

Nucleic Acid Immunogenic Compositions

The present disclosure provides nucleic acid compositions comprising: a)one or more nucleic acids comprising a nucleotide sequence(s) encodingpolypeptides (e.g., HCV E1/E2; HCV E1; HCV E2; T-cell epitopepolypeptide) as described above; and b) a CDN. The present disclosureprovides nucleic acid compositions comprising: a) one or more nucleicacids comprising a nucleotide sequence(s) encoding polypeptides (e.g.,HCV E1/E2; HCV E1; HCV E2; T-cell epitope polypeptide) as describedabove; and b) an archaeosome. The present disclosure provides animmunogenic composition comprising: a) a nucleic acid (e.g., arecombinant viral expression vector(s)) comprising nucleotidesequence(s) encoding one or more of: an HCV E1/E2 heterodimer; an HCV E1polypeptide; an HCV E2 polypeptide; and a T-cell epitope polypeptide,where such polypeptides are described above; and b) a CDN. The presentdisclosure provides an immunogenic composition comprising: a) a nucleicacid (e.g., a recombinant viral expression vector(s)) comprisingnucleotide sequence(s) encoding one or more of: an HCV E1/E2heterodimer; an HCV E1 polypeptide; an HCV E2 polypeptide; and a T-cellepitope polypeptide, where such polypeptides are described above; and b)an archaeosome. The polypeptides can be encoded in the same nucleicacid, or on separate nucleic acids. For example, where the nucleicacid(s) are recombinant expression vectors, the polypeptides can beencoded in the same or separate recombinant expression vectors.

In some cases, the nucleic acid(s) is/are DNA. In some cases, thenucleic acid(s) is/are RNA. In some cases, the nucleic acid(s) is/arepresent in expression vector(s), generating recombinant expressionvector(s) comprising the nucleic acid(s). In some cases, the recombinantexpression vector(s) is/are recombinant bacterial vectors. In somecases, the recombinant expression vector(s) is/are recombinant viralvector(s). In some cases, the recombinant viral vector(s) are packagedinto viral particles. In some cases, the nucleic acid(s) are present inbacteria (e.g., non-pathogenic bacteria (e.g., attenuated bacteria)suitable for delivery of nucleic acids to an individual). Where therecombinant expression vector is a bacterial vector or a viral vector,the vector is suitably attenuated so as not to cause significantpathology in an individual.

In some cases, the nucleic acid is present in an expression vector.Suitable expression vectors include, but are not limited to, areplication-defective adenovirus vector; a replication-defectivevaccinia virus vector; a lentivirus vector (e.g., a self-inactivatinglentivirus vector); a retroviral vector (e.g., a self-inactivatingretroviral vector); an adeno-associated virus vector; and the like. Insome cases, the vector is a modified vaccinia Ankara (MVA) vector, or anMVA-based vector (see, e.g., Verheust et al. (2012) Vaccine 30:2623). Insome cases, the vector is a replication-defective adenovirus vector. Insome cases, the vector is a replication-defective adenovirus 6 (Ad6)vector. In some cases, the vector is a replication-defective simianadenovirus vector (e.g., ChAd3). Suitable viral vectors are describedin, e.g., Zhou et al. (2012) Invest. Ophthalmol. Vis. Sci. 53:2804;Swadling et al. (2014) Sci. Transl. Med. 6:261ra153; and Choi and Chang(2013) Clin. Exp. Vaccine Res. 2:97. In many cases, the recombinantviral vectors are packaged into viral particles; and the viral particlesare formulated in an immunogenic composition along with apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers are described above.

In some cases, an immunogenic composition of the present disclosurecomprises: a) a recombinant viral vector comprising nucleotide sequencesencoding one or more of: an HCV E1/E2 heterodimer; an HCV E1polypeptide; an HCV E2 polypeptide; and a T-cell epitope polypeptide;and b) a CDN or an archaeosome. In some cases, an immunogeniccomposition of the present disclosure comprises: a) a recombinant viralvector comprising nucleotide sequences encoding: an HCV E1/E2heterodimer; and a T-cell epitope polypeptide; and b) a CDN or anarchaeosome. In some cases, an immunogenic composition of the presentdisclosure comprises: a) a recombinant viral vector comprisingnucleotide sequences encoding: an HCV E1 polypeptide; and a T-cellepitope polypeptide; and b) a CDN or an archaeosome. In some cases, animmunogenic composition of the present disclosure comprises: a) arecombinant viral vector comprising nucleotide sequences encoding: anHCV E2 polypeptide; and a T-cell epitope polypeptide; and b) a CDN or anarchaeosome. In some cases, an immunogenic composition of the presentdisclosure comprises: a) a recombinant viral vector comprisingnucleotide sequences encoding an HCV E1/E2 heterodimer; and b) a CDN oran archaeosome. In some cases, an immunogenic composition of the presentdisclosure comprises: a) a recombinant viral vector comprisingnucleotide sequences encoding an HCV E2 polypeptide; and b) a CDN or anarchaeosome. In some cases, an immunogenic composition of the presentdisclosure comprises: a) a recombinant viral vector comprisingnucleotide sequences encoding an HCV E1 polypeptide; and b) a CDN or anarchaeosome.

In some cases, the present disclosure provides: a) a first immunogeniccomposition comprising: i) a recombinant viral vector comprisingnucleotide sequences encoding one or more of: an HCV E1/E2 heterodimer;an HCV E1 polypeptide; an HCV E2 polypeptide; and a T-cell epitopepolypeptide; and ii) a CDN; and b) a second immunogenic compositioncomprising: i) a recombinant viral vector comprising nucleotidesequences encoding one or more of: an HCV E1/E2 heterodimer; an HCV E1polypeptide; an HCV E2 polypeptide; and a T-cell epitope polypeptide;and ii) a CDN. In some cases, the present disclosure provides: a) afirst immunogenic composition comprising: i) a recombinant viral vectorcomprising nucleotide sequences encoding one or more of: an HCV E1/E2heterodimer; an HCV E1 polypeptide; an HCV E2 polypeptide; and a T-cellepitope polypeptide; and ii) an archaeosome; and b) a second immunogeniccomposition comprising: i) a recombinant viral vector comprisingnucleotide sequences encoding one or more of: an HCV E1/E2 heterodimer;an HCV E1 polypeptide; an HCV E2 polypeptide; and a T-cell epitopepolypeptide; and ii) an archaeosome.

In some cases, the present disclosure provides: a) a first immunogeniccomposition comprising: i) a first recombinant viral vector comprisingnucleotide sequences encoding one or more of: an HCV E1/E2 heterodimer;an HCV E1 polypeptide; an HCV E2 polypeptide; and a T-cell epitopepolypeptide; and ii) a CDN or an archaeosome; and b) a secondimmunogenic composition comprising: i) a second recombinant viral vectorcomprising nucleotide sequences encoding one or more of: an HCV E1/E2heterodimer; an HCV E1 polypeptide; an HCV E2 polypeptide; and a T-cellepitope polypeptide; and ii) a CDN or an archaeosome. In some cases, thefirst recombinant viral vector is a replication-defectiveadenovirus-based recombinant viral vector; and the second recombinantviral vector is an MVA-based recombinant viral vector. In some cases,the first recombinant viral vector is a chimpanzee adenovirus-basedrecombinant viral vector; and the second recombinant viral vector is anMVA-based recombinant viral vector.

In some cases, the nucleic acid(s) are present in bacteria (e.g.,non-pathogenic bacteria suitable for delivery of nucleic acids to anindividual). In some cases, the nucleic acid(s) are present inrecombinant expression vector(s) present in bacteria (e.g.,non-pathogenic bacteria suitable for delivery of nucleic acids to anindividual). Thus, the present disclosure provides an immunogeniccomposition comprising a non-pathogenic, bacterium that harbors anucleic acid(s) comprising nucleotide sequences encoding one or more of:an HCV E1/E2 heterodimer; an HCV E1 polypeptide; an HCV E2 polypeptide;and a T-cell epitope polypeptide, where such polypeptides are describedabove. The present disclosure provides an immunogenic compositioncomprising a non-pathogenic bacterium that harbors a recombinantexpression vector(s) comprising nucleotide sequences encoding one ormore of: an HCV E1/E2 heterodimer; an HCV E1 polypeptide; an HCV E2polypeptide; and a T-cell epitope polypeptide, where such polypeptidesare described above. In some cases, the bacteria are live. In somecases, the bacteria are live attenuated bacteria. In some cases, thebacteria are killed.

Bacteria suitable for delivery of nucleic acid(s) (which nucleic acid(s)may be present in expression vector(s)) include, but are not limited to,Lactobacillus; Lactococcus (e.g., Lactococcus lactis); Salmonella, e.g.,attenuated, non-pathogenic Salmonella, e.g., Salmonella enterica serovartyphi, Salmonella enterica serovar typhimurium; non-pathogenic strainsof Francisella; non-pathogenic strains of Escherichia coli;non-pathogenic strains of Bordetella pertussis; non-pathogenic strainsof Listeria; non-pathogenic strains of Shigella; non-pathogenic strainsof Vibrio (e.g., Vibrio cholera); Streptococcus gordonii; non-pathogenicstrains of Yersinia enterocolitica; non-pathogenic strains of Shigellaflexneri; non-pathogenic strains of Pseudomonas aeruginosa;non-pathogenic strains of Bacillus subtilis; and the like.

In some cases, one or more virulence genes in the bacterium is all orpartially deleted. For example, for Salmonella enterica serovar typhiand Salmonella enterica serovar typhimurium, an aroA, aroC, and aroDmutation can be made. Other mutations that can attenuate pathogenicityaffect biosynthesis of the nucleotides adenine (pur) and guanine(guaBA), and outer membrane proteins C and F (ompC, ompF), as well asexpression of the cAMP receptor (cyal crp), the conversion ofUDP-galactose to UDP-glucose (galE), DNA recombination and repair (recA,recBC), and regulation of virulence genes (phoP, phoQ). For Listeriamonocytogenes, attenuation can be achieved with auxotrophic mutants, ordeletion of virulence factors such as the genes actA and internalin B(intB).

Methods of Inducing an Immune Response to HCV

The present disclosure provides a method of inducing an immune response(e.g., a protective immune response) to at least one HCV genotype in amammalian subject (e.g., a human). In some cases, a method of thepresent disclosure for inducing an immune response in an individual toat least one HCV genotype comprises administering an immunogeniccomposition of the present disclosure, where the immunogenic compositioncomprises polypeptides (e.g., HCV E1/E2, HCV E1, or HCV E2; andoptionally a T-cell epitope polypeptide). In some cases, a method of thepresent disclosure for inducing an immune response in an individual toat least one HCV genotype comprises administering an immunogeniccomposition of the present disclosure, where the immunogenic compositioncomprises one or more nucleic acids comprising nucleotide sequencesencoding polypeptides (e.g., e.g., HCV E1/E2, HCV E1, or HCV E2; andoptionally a T-cell epitope polypeptide).

Administering an Immunogenic Composition Comprising Polypeptides

In some cases, the methods comprise administering to an individual inneed thereof an effective amount of an immunogenic composition of thepresent disclosure, where the immunogenic composition comprises: a) anHCV E1/E2 heterodimer; and b) a CDN; or where the immunogeniccomposition comprises: a) an HCV E2 polypeptide; and b) a CDN; or wherethe immunogenic composition comprises: a) an HCV E1 polypeptide; and b)a CDN. In some cases, the methods comprise administering to anindividual in need thereof an effective amount of an immunogeniccomposition of the present disclosure, where the immunogenic compositioncomprises: a) an HCV E1/E2 heterodimer; and b) an archaeosome; or wherethe immunogenic composition comprises: a) an HCV E2 polypeptide; and b)an archaeosome; or where the immunogenic composition comprises: a) anHCV E1 polypeptide; and b) an archaeosome.

In some cases, the methods comprise administering to an individual inneed thereof an effective amount of an immunogenic composition of thepresent disclosure, where the immunogenic composition comprises: a) anHCV E1/E2 heterodimer; b) a T-cell epitope polypeptide comprising aT-cell epitope present in an HCV polypeptide other than E1 and E2; andc) a CDN; or where the immunogenic composition comprises: a) an HCV E2polypeptide; b) a T-cell epitope polypeptide comprising a T-cell epitopepresent in an HCV polypeptide other than E1 and E2; and c) a CDN; orwhere the immunogenic composition comprises: a) an HCV E1 polypeptide;b) a T-cell epitope polypeptide comprising a T-cell epitope present inan HCV polypeptide other than E1 and E2; and c) a CDN.

In some cases, the methods comprise administering to an individual inneed thereof an effective amount of an immunogenic composition of thepresent disclosure, where the immunogenic composition comprises: a) anHCV E1/E2 heterodimer; b) a T-cell epitope polypeptide comprising aT-cell epitope present in an HCV polypeptide other than E1 and E2; andc) an archaeosome; or where the immunogenic composition comprises: a) anHCV E2 polypeptide; b) a T-cell epitope polypeptide comprising a T-cellepitope present in an HCV polypeptide other than E1 and E2; and c) anarchaeosome; or where the immunogenic composition comprises: a) an HCVE1 polypeptide; b) a T-cell epitope polypeptide comprising a T-cellepitope present in an HCV polypeptide other than E1 and E2; and c) anarchaeosome.

In some cases, an immunogenic composition of the present disclosurecomprising an HCV E1/E2 heterodimer and a CDN is administered viaintramuscular administration. In some cases, an immunogenic compositionof the present disclosure comprising an HCV E1/E2 heterodimer and a CDNis administered via intranasal administration. In some cases, animmunogenic composition of the present disclosure comprising an HCVE1/E2 heterodimer and a CDN is administered: a) first via intramuscularadministration; b) followed by a second administration via intranasaladministration; c) followed by a third administration via intranasaladministration.

In some cases, an immunogenic composition of the present disclosurecomprising an HCV E1/E2 heterodimer and an archaeosome is administeredvia intramuscular administration.

Administering an Immunogenic Composition Comprising Nucleic Acid(s)

In some cases, a method of the present disclosure for inducing an immuneresponse to HCV in an individual comprises administering to theindividual an effective amount of an immunogenic composition comprising:a) nucleic acid(s) comprising nucleotide sequences encoding: 1) an HCVE1/E2 heterodimer and a T-cell epitope polypeptide; 2) an HCV E2polypeptide and a T-cell epitope polypeptide; 3) an HCV E1 polypeptideand a T-cell epitope polypeptide; 4) an HCV E1/E2 heterodimer; 5) an HCVE2 polypeptide; or 6) an HCV E1 polypeptide; and b) a CDN or anarchaeosome. The polypeptides can be encoded in the same nucleic acid,or on separate nucleic acids. For example, where the nucleic acid(s) arerecombinant expression vectors, the polypeptides can be encoded in thesame or separate recombinant expression vectors.

In some cases, the nucleic acid(s) is/are DNA. In some cases, thenucleic acid(s) is/are RNA. In some cases, the nucleic acid(s) is/arepresent in expression vector(s) such that a recombinant expressionvector(s) comprising the nucleic acid(s) are administered. In somecases, the recombinant expression vector(s) is/are recombinant viralvector(s). In some cases, the recombinant viral vector(s) are packagedinto viral particles. In some cases, the nucleic acid(s) are present inbacteria (e.g., non-pathogenic bacteria (e.g., attenuated bacteria)suitable for delivery of nucleic acids to an individual).

In some cases, the nucleic acid is present in an expression vector,thereby generating a recombinant expression vector. Suitable expressionvectors include, but are not limited to, a replication-defectiveadenovirus vector; a replication-defective vaccinia virus vector; alentivirus vector (e.g., a self-inactivating lentivirus vector); aretroviral vector (e.g., a self-inactivating retroviral vector); anadeno-associated virus vector; and the like. In some cases, the vectoris a modified vaccinia Ankara (MVA) vector, or an MVA-based vector (see,e.g., Verheust et al. (2012) Vaccine 30:2623). In some cases, the vectoris a replication-defective adenovirus vector. In some cases, the vectoris a replication-defective adenovirus 6 (Ad6) vector. In some cases, thevector is a replication-defective simian adenovirus vector (e.g.,ChAd3). Suitable viral vectors are described in, e.g., Zhou et al.(2012) Invest. Ophthalmol. Vis. Sci. 53:2804; Swadling et al. (2014)Sci. Transl. Med. 6:261ra153; and Choi and Chang (2013) Clin. Exp.Vaccine Res. 2:97. In many cases, the recombinant viral vectors arepackaged into viral particles; and the viral particles are formulated inan immunogenic composition along with a pharmaceutically acceptablecarrier.

In some cases, an HCV E1/E2 heterodimer is encoded by nucleotidesequences present in a first recombinant viral vector, e.g., anadenovirus vector, a vaccinia virus vector, an MVA vector or MVA-basedvector; and a T-cell epitope polypeptide is encoded by nucleotidesequences present in a second recombinant viral vector, e.g., anadenovirus vector, a vaccinia virus vector, an MVA vector or MVA-basedvector.

In some cases, a prime-boost vaccine protocol is used. In some cases, afirst (priming) immunogenic composition is administered, where the firstimmunogenic composition comprises a recombinant viral vector comprisingnucleotide sequences encoding one or more of: a) an HCV E1/E2heterodimer; b) an HCV E2 polypeptide; c) an HCV E1 polypeptide; and d)a T-cell epitope polypeptide; and, after a time, a second (booster)immunogenic composition is administered, where the second immunogeniccomposition comprises a recombinant viral vector comprising nucleotidesequences encoding one or more of: a) an HCV E1/E2 heterodimer; b) anHCV E2 polypeptide; c) an HCV E1 polypeptide; and d) a T-cell epitopepolypeptide. In some cases, the first recombinant viral vector and thesecond recombinant viral vector are the same. In some cases, the firstrecombinant viral vector and the second recombinant viral vector aredifferent. For example, in some cases, the first recombinant viralvector is a vaccinia-based recombinant viral vector; and the secondrecombinant viral vector is an adenovirus-based recombinant viralvector. In general, the recombinant viral vectors are packaged intoviral particles. A second immunogenic composition can be administered ata time period of from 1 day to 1 year following administration of thefirst immunogenic composition. For example, a second immunogeniccomposition can be administered at a time period of from 1 day to 1week, from 1 week to 2 weeks, from 2 weeks to 1 month, from 1 month to 2months, from 2 months to 6 months, or from 6 months to 1 year followingadministration of the first immunogenic composition.

For example, in some cases, a first (priming) vaccine comprising arecombinant adenovirus (e.g., Ad6 or chimpanzee Ad (e.g., ChAd3)) thatcomprises a nucleotide sequence encoding an HCV E1/E2 heterodimer isfollowed by a second (booster) vaccine comprising a recombinant MVAvector that comprises a nucleotide sequence encoding a T-cell epitopepolypeptide. Other prime-boost protocols can be used. For example,multiple primes and/or multiple boosts can be administered.

In some cases, a first (priming) immunogenic composition isadministered, where the first immunogenic composition comprises one ormore of: a) an HCV E1/E2 heterodimer; b) an HCV E2 polypeptide; c) anHCV E1 polypeptide; and d) a T-cell epitope polypeptide, as describedabove; and a second (boosting) immunogenic composition is administered,where the second immunogenic composition comprises a recombinant viralvector comprising nucleotide sequence(s) encoding one or more of: a) anHCV E1/E2 heterodimer; b) an HCV E2 polypeptide; c) an HCV E1polypeptide; and d) a T-cell epitope polypeptide, as described above.

In some cases, a first (priming) immunogenic composition isadministered, where the first immunogenic composition comprises arecombinant viral vector comprising nucleotide sequence(s) encoding oneor more of: a) an HCV E1/E2 heterodimer; b) an HCV E2 polypeptide; c) anHCV E1 polypeptide; and d) a T-cell epitope polypeptide, as describedabove; and a second (boosting) immunogenic composition is administered,where the second immunogenic composition comprises one or more of: a) anHCV E1/E2 heterodimer; b) an HCV E2 polypeptide; c) an HCV E1polypeptide; and d) a T-cell epitope polypeptide, as described above.

In some cases, a co-immunization regimen is carried out, in which apolypeptide(s) per se is administered substantially concomitantly with anucleic acid(s) encoding the polypeptide(s). For example, in some cases,a method of the present disclosure for inducing an immune response to anHCV polypeptide can comprise administering: a) a first immunogeniccomposition of the present disclosure, as described above, where theimmunogenic composition comprises: i) an HCV E1/E2 heterodimer; ii) aT-cell epitope polypeptide; and iii) a pharmaceutically acceptablecarrier; or i) an HCV E1 polypeptide; ii) a T-cell epitope polypeptide;and iii) a pharmaceutically acceptable carrier; or i) an HCV E2polypeptide; ii) a T-cell epitope polypeptide; and iii) apharmaceutically acceptable carrier; and b) a second immunogeniccomposition of the present disclosure, as described above, where theimmunogenic composition comprises: i) one or more nucleic acidscomprising nucleotide sequence encoding one or more of: an HCV E1/E2heterodimer, an HCV E1 polypeptide, an HCV E2 polypeptide, and a T-cellepitope polypeptide; and ii) a pharmaceutically acceptable carrier. Insome cases, the first and the second immunogenic compositions are in asingle formulation. In some cases, the first and the second immunogeniccompositions are in separate formulations. In some cases, the first andthe second immunogenic compositions are administered via the same routeof administration. In some cases, the first and the second immunogeniccompositions are administered via different routes of administration. Insome cases, the first and the second immunogenic compositions are inseparate formulations that are administered substantiallysimultaneously, e.g., within 1 minute, within 1 minute to 5 minutes,within 5 minutes to 15 minutes, or within 15 minutes to 30 minutes, ofone another. In some cases, the first and the second immunogeniccompositions are administered multiple times to an individual.

In some cases, a co-immunization regimen is carried out, in which apolypeptide(s) per se is administered substantially concomitantly with anucleic acid(s) encoding the polypeptide(s). For example, in some cases,a method of the present disclosure for inducing an immune response to anHCV polypeptide can comprise administering: a) a first immunogeniccomposition of the present disclosure, as described above, where theimmunogenic composition comprises: i) an HCV E1/E2 heterodimer; ii) aT-cell epitope polypeptide; and iii) a CDN; and b) a second immunogeniccomposition of the present disclosure, as described above, where theimmunogenic composition comprises: i) one or more nucleic acidscomprising nucleotide sequences encoding an HCV E1/E2 heterodimer and aT-cell epitope polypeptide; and ii) a CDN. In some cases, the first andthe second immunogenic compositions are in a single formulation. In somecases, the first and the second immunogenic compositions are in separateformulations. In some cases, the first and the second immunogeniccompositions are administered via the same route of administration. Insome cases, the first and the second immunogenic compositions areadministered via different routes of administration. In some cases, thefirst and the second immunogenic compositions are in separateformulations that are administered substantially simultaneously, e.g.,within 1 minute, within 1 minute to 5 minutes, within 5 minutes to 15minutes, or within 15 minutes to 30 minutes, of one another. In somecases, the first and the second immunogenic compositions areadministered multiple times to an individual. In some cases, the one ormore nucleic acids are recombinant viral vectors.

In some cases, a method of the present disclosure for inducing an immuneresponse to HCV in an individual comprises administering to theindividual an effective amount of an immunogenic composition comprising:a) nucleic acid(s) comprising nucleotide sequences encoding 1) an HCVE1/E2 heterodimer and a T-cell epitope polypeptide; 2) an HCV E2polypeptide and a T-cell epitope polypeptide; 3) an HCV E1 polypeptideand a T-cell epitope polypeptide; 4) an HCV E1/E2 polypeptide; 5) an HCVE2 polypeptide; or 6) an HCV E1 polypeptide; and b) a CDN or anarchaeosome. In some cases, the nucleic acid is an RNA comprisingnucleotide sequences encoding a polypeptide of the present disclosure(e.g., an HCV E1/E2 heterodimer; an HCV E1 polypeptide; an HCV E2polypeptide; a T-cell epitope polypeptide, as described herein. See,e.g., Weiner (2013) Molec. Therapy 21:506; and Ulmer et al. (2012)Vaccine 30:4414. In some cases, an RNA (e.g., a single mRNA molecule; or2 mRNA molecules; or 3 mRNA molecules) comprising nucleotide sequencesencoding a polypeptide of the present disclosure is formulated with aliposome. In some cases, an RNA (e.g., a single mRNA molecule; or 2 mRNAmolecules) comprising nucleotide sequences encoding a polypeptide of thepresent disclosure is complexed with protamine. In some cases, an RNA(e.g., a single mRNA molecule; or 2 mRNA molecules) comprisingnucleotide sequences encoding a polypeptide of the present disclosure iscomplexed with1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOTAP/DOPE).

In some cases, the nucleic acid(s) are present in bacteria (e.g.,non-pathogenic bacteria suitable for delivery of nucleic acids to anindividual). In some cases, the nucleic acid(s) are present inrecombinant expression vector(s) present in bacteria (e.g.,non-pathogenic bacteria suitable for delivery of nucleic acids to anindividual). In some cases, the bacteria are live. In some cases, thebacteria are live attenuated bacteria. In some cases, the bacteria arekilled. Bacteria suitable for delivery of nucleic acid(s) (which may bepresent in expression vectors) include, but are not limited to,Lactobacillus; Lactococcus (e.g., Lactococcus lactis); Salmonella, e.g.,attenuated, non-pathogenic Salmonella, e.g., Salmonella enterica serovartyphi, Salmonella enterica serovar typhimurium; non-pathogenic strainsof Escherichia coli; non-pathogenic strains of Bordetella pertussis;non-pathogenic strains of Listeria; non-pathogenic strains of Shigella;non-pathogenic strains of Vibrio (e.g., Vibrio cholera); Streptococcusgordonii; non-pathogenic strains of Yersinia enterocolitica;non-pathogenic strains of Shigella flexneri; non-pathogenic strains ofPseudomonas aeruginosa; non-pathogenic strains of Bacillus subtilis; andthe like. In some cases, one or more virulence genes in the bacterium isall or partially deleted. For example, for Salmonella enterica serovartyphi and Salmonella enterica serovar typhimurium, an aroA, aroC, andaroD mutation can be made. Other mutations that can attenuatepathogenicity affect biosynthesis of the nucleotides adenine (pur) andguanine (guaBA), and outer membrane proteins C and F (ompC, ompF), aswell as expression of the cAMP receptor (cya/crp), the conversion ofUDP-galactose to UDP-glucose (galE), DNA recombination and repair (recA,recBC), and regulation of virulence genes (phoP, phoQ). For Listeriamonocytogenes, attenuation can be achieved with auxotrophic mutants, ordeletion of virulence factors such as the genes actA and internalin B(intB).

General Considerations

An immunogenic composition of the present disclosure is generallyadministered to a human subject who has an HCV infection or who is atrisk of acquiring an HCV infection (e.g., is at greater risk than thegeneral population of acquiring an HCV infection) so as to prevent or atleast partially arrest the development of disease and its complications.An amount adequate to accomplish this is defined as a “therapeuticallyeffective dose” or a “therapeutically effective amount.” “Prophylactic”use of a subject immunogenic composition generally refers toadministration to an individual who has not been infected with HCV.“Therapeutic” use of a subject immunogenic composition can refer to“prophylactic” use (administration to an individual who has not beeninfected with HCV) and/or to administration to an individual who has anHCV infection. A “therapeutically effective amount” of an immunogeniccomposition of the present disclosure, can be an amount that, whenadministered in one or more doses to an individual who is not infectedwith HCV, is effective to induce an immune response in the individual toHCV. A “therapeutically effective amount” of an immunogenic compositionof the present disclosure, can be an amount that, when administered inone or more doses to an individual who is infected with HCV, iseffective to enhance an immune response in the individual to HCV.

Amounts effective for therapeutic use will depend on, e.g., the mannerof administration, the weight and general state of health of thepatient, and the judgment of the prescribing physician. Single ormultiple doses of a subject immunogenic composition can be administereddepending on the dosage and frequency required and tolerated by thepatient, and route of administration.

In some cases, an effective amount of an immunogenic composition of thepresent disclosure is an amount that, when administered to an individualin one or more doses, is effective to induce an antibody response (e.g.,a neutralizing antibody response) to HCV in the individual. For example,antibody to HCV (e.g., extracellular HCV), and/or to an HCV-infectedcell, can be induced.

An effective amount of an immunogenic composition of the presentdisclosure can be an amount that, when administered to an individual inone or more doses, is effective to induce a neutralizing antibodyresponse to HCV of a variety of genotypes (e.g., genotype 1; genotype 3;etc.). A neutralizing antibody response reduces binding of HCV to one ormore host receptors for HCV and inhibits entry of HCV into a cell.

In some cases, an effective amount (e.g., a therapeutically effectiveamount) of an immunogenic composition of the present disclosure is anamount that, when administered to an individual in one or more doses, iseffective to induce a cytotoxic T lymphocyte (CTL) response to HCV. Forexample, a CTL response to an HCV-infected cell can be induced.

In some cases, an effective amount (e.g., a therapeutically effectiveamount) of an immunogenic composition of the present disclosure is anamount that, when administered to an individual in one or more doses, iseffective to induce a helper T lymphocyte (e.g., CD4⁺ T cell) to HCV inan individual.

In some cases, an effective amount (e.g., a therapeutically effectiveamount) of an immunogenic composition of the present disclosure is anamount that, when administered to an individual in one or more doses, iseffective to induce an antibody response (e.g., a neutralizing antibodyresponse) and/or a CTL response and/or a helper T cell response to HCVgenotype 1. In some cases, an effective amount (e.g., a therapeuticallyeffective amount) of an immunogenic composition of the presentdisclosure is an amount that, when administered to an individual in oneor more doses, is effective to induce an antibody response (e.g., aneutralizing antibody response) and/or a CTL response and/or a helper Tcell response to HCV genotype 3. In some cases, an effective amount(e.g., a therapeutically effective amount) of an immunogenic compositionof the present disclosure is an amount that, when administered to anindividual in one or more doses, is effective to induce an antibodyresponse (e.g., a neutralizing antibody response) and/or a CTL responseand/or a helper T cell response to HCV genotype 1 and HCV genotype 3. Insome cases, an effective amount (e.g., a therapeutically effectiveamount) of an immunogenic composition of the present disclosure is anamount that, when administered to an individual in one or more doses, iseffective to induce an antibody response (e.g., a neutralizing antibodyresponse) and/or a CTL response and/or a helper T cell response to HCVof any genotype.

An immunogenic composition of the present disclosure is generallyadministered in an amount effective to elicit an immune response, e.g.,a humoral immune response (e.g., an antibody response) and/or a CTLresponse, in the mammalian subject. Effective amounts for immunizationwill vary, and can generally range from about 1 μg to 100 μg per 70 kgpatient, e.g., from about 5 μg/70 kg to about 50 μg/70 kg. Substantiallyhigher dosages (e.g. 10 mg to 100 mg or more) may be suitable in oral,nasal, or topical administration routes. The initial administration canbe followed by booster immunization of the same immunogenic compositionor a different immunogenic composition. In some instances, a subjectmethod of inducing an immune response involves an initial administrationof an immunogenic composition of the present disclosure, followed by atleast one booster, and in some instances involves two or more (e.g.,three, four, or five) boosters. The interval between an initialadministration and a booster, or between a give booster and a subsequentbooster, can be from about 1 week to about 12 weeks, e.g., from about 1week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about8 weeks to about 10 weeks, or from about 10 weeks to about 12 weeks. Theinterval between an initial administration and a booster, or between agive booster and a subsequent booster, can be from 4 months to 6 months,or from 6 months to 1 year.

In general, immunization can be accomplished by administration of animmunogenic composition of the present disclosure by any suitable route,including administration of the composition orally, nasally,nasopharyngeally, parenterally, enterically, gastrically, topically,transdermally, subcutaneously, intramuscularly, in tablet, solid,powdered, liquid, aerosol form, locally or systemically, with or withoutadded excipients. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 15th ed., Mack Publishing Company,Easton, Pa. (1980). In some instances, immunization is accomplished byintramuscular injection of an immunogenic composition of the presentdisclosure.

Individuals Suitable for Administration

Individuals who are suitable for administration with an immunogeniccomposition of the present disclosure include immunologically naïveindividuals (e.g., individuals who have not been infected with HCVand/or who have not been administered with an HCV vaccine). Individualssuitable for administration include humans. Individuals who are suitablefor administration with an immunogenic composition of the presentdisclosure are also referred to as “an individual in need thereof.”

Individuals who are suitable for administration with an immunogeniccomposition of the present disclosure composition of the presentdisclosure include individuals who are at greater risk than the generalpopulation of becoming infected with HCV, where such individualsinclude, e.g., intravenous drug users; individuals who are therecipients, or the prospective recipients, of blood or blood productsfrom another (donor) individual(s); individuals who are the recipients,or the prospective recipients, of non-autologous cells, tissues, ororgans from another (donor) individual; health care workers; emergencymedical and non-medical personnel (e.g., first responders; firefighters; emergency medical team personnel; etc.) and the like.

Individuals who are suitable for administration with an immunogeniccomposition of the present disclosure composition of the presentdisclosure include individuals who recently became exposed to HCV or whorecently became infected with HCV. For example, a subject immunogeniccomposition can be administered to an individual within from about 24hours to about 48 hours, from about 48 hours to about 1 week, or fromabout 1 week to about 4 weeks, following possible or suspected exposureto HCV or following infection with HCV.

Individuals who are suitable for administration with an immunogeniccomposition of the present disclosure composition of the presentdisclosure include individuals who have been diagnosed as having an HCVinfection, and include chronically infected individuals. In some cases,an individual who has been diagnosed as having an HCV infection istreated with an anti-viral agent and an immunogenic composition of thepresent disclosure. Suitable anti-viral agents for treating HCVinfection include, e.g., ribavirin(1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide); interferon-alpha(IFN-α) (where “IFN-α” includes IFN-α2a; IFN-α2b; IFN-α that isconjugated with poly(ethylene glycol) (“pegylated IFN-α), where thepegylated IFN-α can be pegylated IFN-α2a or pegylated IFN-α 2b); an HCVNS3 protease inhibitor (e.g., boceprevir; telaprevir); and an HCV NS5protease inhibitor. In some cases, an individual who has been diagnosedas having an HCV infection is treated with, e.g.: 1) IFN-α+ribavirin;and an immunogenic composition of the present disclosure; or 2)IFN-α+ribavirin+an HCV protease inhibitor (e.g., boceprevir ortelaprevir); and an immunogenic composition of the present disclosure.Suitable anti-viral agents for treating HCV infection include Sovaldi(Sofosbuvir; a nucleotide analog that functions as an NS5B polymeraseinhibitor), alone or in combination with pegylated IFN-α and ribavirin.

Examples of Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-136 areprovided below. As will be apparent to those of skill in the art uponreading this disclosure, each of the individually numbered aspects maybe used or combined with any of the preceding or following individuallynumbered aspects. This is intended to provide support for all suchcombinations of aspects and is not limited to combinations of aspectsexplicitly provided below:

Aspect 1. An immunogenic composition comprising: a) an HCV E1/E2heterodimer; and b) a cyclic dinucleotide (CDN); or a) an HCV E2polypeptide; and b) a CDN; or a) an HCV E1 polypeptide; and b) a CDN.

Aspect 2. The immunogenic composition of aspect 1A, aspect 1B, or aspect1C, wherein the CDN is fluorinated.

Aspect 3. The immunogenic composition of aspect 2, wherein the CDN is2′-F-c-di-GMP.

Aspect 4. The immunogenic composition of aspect 1A, aspect 1B, or aspect1C, wherein the CDN is of Formula (I):

wherein:

A is S or O;

X is S, N, O, CH₂;

Y, Y′ is NH, CH₂, O;

Z, Z′ is NH, CH₂, O;

R1 represents hydrogen or NH₂ which may be substituted;

R2 is hydrogen or absent;

R3 represents NH₂, O, OH, H, or a halogen;

R4 represents hydrogen, halogen, or a straight or branched C₁-C₆ alkylgroup which may optionally be substituted;

R5 represents hydrogen, OH or a straight or branched C₁-C₆ alkyl chainor C₁-C₆ straight or branched alkoxy chain which may optionally besubstituted;

is a single or double bond;

or conjugates thereof, and salts or solvates thereof.

Aspect 5. The immunogenic composition of aspect 4, wherein the CDN isc-diGMP, c-diAMP, c-dilMP, or c-dXMP.

Aspect 6. The immunogenic composition of aspect 4, wherein the CDN isc-GpAp, c-Gplp, c-GpXp, c-Aplp, c-ApXp, or c-lpXp.

Aspect 7. The immunogenic composition of aspect 1, wherein the CDN iscyclic-GMP-AMP (cGAMP).

Aspect 8. The immunogenic composition of aspect 7, wherein the cGAMP is2′3′-cGAMP, 2′2-cGAMP, 3′2′-cGAMP or 3′3′-GAMP.

Aspect 9. The immunogenic composition of any one of aspect aspects 1-8,wherein the composition comprises an HCV E1/E2 heterodimer.

Aspect 10. The immunogenic composition of aspect 9, wherein:

a) the HCV E2 polypeptide is derived from an HCV of major genotype 1, 2,3, 4, 5, 6, or 7; and

b) the HCV E1 polypeptide is derived from an HCV of major genotype 1, 2,3, 4, 5, 6, or 7.

Aspect 11. The immunogenic composition of any one of aspects 1-10,wherein the HCV E2 polypeptide comprises an amino acid sequence havingat least 20% amino acid sequence identity to an E2 polypeptide depictedin one of FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B.

Aspect 12. The immunogenic composition of any one of aspects 1-10,wherein the HCV E1 polypeptide comprises an amino acid sequence havingat least 20% amino acid sequence identity to an E1 polypeptide depictedin FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B.

Aspect 13. The immunogenic composition of any one of aspects 1-12,wherein the E2 polypeptide and/or the E1 polypeptide lacks a C-terminaltransmembrane domain.

Aspect 14. The immunogenic composition of any one of aspects 1-13,wherein the HCV E2 polypeptide and the HCV E1 polypeptide are derivedfrom an HCV of the same genotype.

Aspect 15. The immunogenic composition of any one of aspects 1-13,wherein the HCV E2 polypeptide and the HCV E1 polypeptide are derivedfrom an HCV of different genotypes.

Aspect 16. The immunogenic composition of any one of aspects 1-15,wherein the HCV E1/E2 heterodimeric polypeptide comprises:

a) an HCV E1 polypeptide; and

b) a modified E2 polypeptide comprising, in order from N-terminus toC-terminus:

i) from 1 to 6 heterologous amino acids, wherein the from 1 to 6heterologous amino acids are C-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; and

ii) an HCV E2 polypeptide; or

a) an HCV E2 polypeptide; and

b) a modified E1 polypeptide comprising, in order from N-terminus toC-terminus:

i) from 1 to 6 heterologous amino acids, wherein the from 1 to 6heterologous amino acids are C-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; and

ii) an HCV E1 polypeptide; or

a) an HCV E1 polypeptide; and

b) a modified E2 polypeptide comprising, in order from N-terminus toC-terminus:

i) an HCV E2 polypeptide; and

ii) from 1 to 6 heterologous amino acids, wherein the from 1 to 6heterologous amino acids are N-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; or

a) an HCV E2 polypeptide; and

b) a modified E1 polypeptide comprising, in order from N-terminus toC-terminus:

-   -   i) an HCV E1 polypeptide; and    -   ii) from 1 to 6 heterologous amino acids, wherein the from 1 to        6 heterologous amino acids are N-terminal to a site of        proteolytic cleavage in a proteolytically cleavable linker.

Aspect 17. The immunogenic composition of aspect 16, wherein:

a) the from 1 to 6 heterologous amino acids at the N-terminus of themodified E2 polypeptide or the modified E1 polypeptide are Gly-Pro, Ser,Gly, or Gly-Ser; or

b) the from 1 to 6 heterologous amino acids at the C-terminus of themodified E2 polypeptide or the modified E1 polypeptide are LEVLFQGP (SEQID NO:117), ENLYYFQ (SEQ ID NO:150), LVPR (SEQ ID NO:124), I(E/D)GR (SEQID NO:125), or DDDDK.

Aspect 18. The immunogenic composition of any one of aspects 1-17,wherein the HCV E1 polypeptide, the HCV E2 polypeptide, or one or bothchains of the HCV E1/E2 heterodimer comprises a covalently linked T-cellepitope polypeptide comprising a T-cell epitope present in an HCVprotein other than E1 and E2.

Aspect 19. The immunogenic composition of any one of aspects 1-18,comprising a T-cell epitope polypeptide comprising a T-cell epitopepresent in an HCV protein other than E1 and E2, wherein the T-cellepitope polypeptide is not covalently linked to the HCV E1E2heterodimer, the HCV E1 polypeptide, or the HCV E2 polypeptide.

Aspect 20. The immunogenic composition of aspect 18 or aspect 19,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in one or more of:

a) an HCV non-structural polypeptide-3 (NS3) polypeptide;

b) an HCV non-structural polypeptide-2 (NS2) polypeptide;

c) an HCV non-structural polypeptide-4A (NS4A) polypeptide;

d) an HCV non-structural polypeptide-4B (NS4B) polypeptide;

e) an HCV non-structural polypeptide-5A (NS5A) polypeptide;

f) an HCV non-structural polypeptide-5B (NS5B) polypeptide;

g) an HCV core polypeptide; and

h) an HCV p7 polypeptide.

Aspect 21. The immunogenic composition of any one of aspects 18-20,wherein the T-cell epitope polypeptide has a length of from about 10amino acids to about 3000 amino acids.

Aspect 22. The immunogenic composition of any one of aspects 18-20,wherein the T-cell epitope polypeptide has a length of from about 10amino acids to about 50 amino acids, from about 100 amino acids to about230 amino acids, from about 230 amino acids to about 550 amino acids,from about 550 amino acids to about 780 amino acids, or from about 780amino acids to about 2000 amino acids.

Aspect 23. The immunogenic composition of any one of aspects 18-22,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in an HCV NS3 polypeptide.

Aspect 24. The immunogenic composition of any one of aspects 18-23,wherein the T-cell epitope polypeptide comprises an amino acid sequencehaving at least 20% amino acid sequence identity to the amino acidsequence of one of TP29, TP50, TP52, TP70, TP100, TP171, TP228, TP553,TP778, and TP1985.

Aspect 25. The immunogenic composition of aspect any one of aspects18-24, wherein the T-cell epitope polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to apolypeptide depicted in any one of FIGS. 9A, 9B, 10A-10D, and 11A-11N.

Aspect 26. The immunogenic composition of any one of 18-25, wherein theT-cell epitope polypeptide comprises one or more T cell epitopes presentin:

a) cholera toxin or toxoid; and/or

b) tetanus toxin or toxoid; and/or

c) diphtheria toxin or toxoid; and/or

d) CRM197.

Aspect 27. The immunogenic composition of any one of aspects 1-25,wherein the composition comprises a polypeptide comprising one or more Tcell epitopes present in:

a) cholera toxin or toxoid; and/or

b) tetanus toxin or toxoid; and/or

c) diphtheria toxin or toxoid; and/or

d) CRM197.

Aspect 28. A method of inducing an immune response to HCV in anindividual, the method comprising administering to the individual aneffective amount of the immunogenic composition of any one of aspects1-27.

Aspect 29. The method of aspect 28, wherein said administering is viaintramuscular administration, intranasal administration, subcutaneousadministration, or a combination thereof.

Aspect 30. The method of aspect 28, wherein said administering comprisesa prime and a boost.

Aspect 31. An immunogenic composition comprising:

a) one or more nucleic acids comprising nucleotide sequences encodingthe hepatitis C virus (HCV) E1 polypeptide, the HCV E2 polypeptide, orthe HCV E1/E2 heterodimer as recited in any one of aspects 1-28; and

b) a cyclic dinucleotide as recited in any one of aspects 1-28.

Aspect 32. The immunogenic composition of aspect 31, wherein the one ormore nucleic acids are recombinant expression vectors.

Aspect 33. The immunogenic composition of aspect 32, wherein the one ormore recombinant expression vectors are recombinant viral vectors.

Aspect 34. The immunogenic composition of aspect 32, wherein the one ormore recombinant viral vectors are packaged into viral particles.

Aspect 35. The immunogenic composition of any one of aspects 31-34,wherein the one or more nucleic acids are present within non-pathogenicbacteria.

Aspect 36. A method of inducing an immune response to HCV in anindividual, the method comprising administering to the individual aneffective amount of any one of aspects 31-35.

Aspect 37. The method of aspect 36, wherein said administering is viaintramuscular administration, intranasal administration, subcutaneousadministration, or a combination thereof.

Aspect 38. The method of aspect 36, wherein said administering comprisesa prime and a boost.

Aspect 39. The method of any one of aspects 28-30 or aspects 36-38,wherein the immune response comprises one or more of a CD4⁺ response, aCD8⁺ response, and a neutralizing antibody response.

Aspect 40. The method of any one of aspects 28-30 or aspects 36-38,wherein the immune response induced is to more than one HCV genotype.

Aspect 41. An immunogenic composition comprising: a) an HCV E1/E2heterodimer; and b) an archaeosome comprising at least one polarsynthetic lipid, wherein the at least one polar synthetic lipidcomprises at least one carbohydrate or anionic group linked by covalentbonding to at least one free hydroxyl group of an archaeal core lipid;or a) an HCV E2 polypeptide; and b) an archaeosome comprising at leastone polar synthetic lipid, wherein the at least one polar syntheticlipid comprises at least one carbohydrate or anionic group linked bycovalent bonding to at least one free hydroxyl group of an archaeal corelipid; or a) an HCV E1 polypeptide; and b) an archaeosome comprising atleast one polar synthetic lipid, wherein the at least one polarsynthetic lipid comprises at least one carbohydrate or anionic grouplinked by covalent bonding to at least one free hydroxyl group of anarchaeal core lipid.

Aspect 42. The immunogenic composition of aspect 41, wherein thearchaeal core lipid is archaeol (2,3-di-O-diphytanyl-sn-glycerol).

Aspect 43. The immunogenic composition of aspect 41, wherein thearchaeal core lipid is caldarchaeol(2,2′,3,3′-tetra-O-dibiphytanyl-sn-diglycerol).

Aspect 44. The immunogenic composition of aspect 41, wherein thecarbohydrate group is selected from the group consisting of:β-D-Glc-(1,6)-β-D-Glc-; β-D-Glc-(1,6)-.alpha.-D-Glc-;α-D-Glc-(1,4)-β-D-Glc-; β-D-Gal-(1,4)-13-D-Glc-; α-D-Gal-(1,6)-β-D-Glc-;β-D-Glc-(1,6)-β-D-Glc-(1,6)-β-D-Glc-;α-D-Glc-(1,4)-α-D-Glc-(1,4)-β-D-Glc-;α-D-Man-(1,2)-α-D-Man-(1,2)-α-D-Man-; andα-D-Man-(1,2)-α-D-Man-(1,2)-α-D-Man-(1,2)-α-D-Man-.

Aspect 45. The immunogenic composition of aspect 41, wherein thecarbohydrate group comprises two or three β-D-Glc-units in (1,6)linkage.

Aspect 46. The immunogenic composition of aspect 41, wherein thecarbohydrate group is a gal-glc-group.

Aspect 47. The immunogenic composition of aspect 41, wherein the anionicgroup is selected from the group consisting of phosphoserine,phosphoethanolamine, phosphoinositol and phosphoglycerol.

Aspect 48. The immunogenic composition of aspect 41, wherein the atleast one polar synthetic lipid comprises at least one anionic lipid.

Aspect 49. The immunogenic composition of aspect 48, wherein the atleast one anionic lipid is selected from the group consisting ofarchaetidylglycerol, archaetidylglycerolphosphate-methyl,archaetidylserine, and archaetidylinositol.

Aspect 50. The immunogenic composition of aspect 48, wherein thearchaeosome comprises at least one conventional lipid.

Aspect 51. The immunogenic composition of aspect 50, wherein the atleast one conventional lipid is selected from a group consisting ofphosphatidylglycerol, phosphatidylserine, SQDG, and cholesterol.

Aspect 52. The immunogenic composition of aspect 51, wherein the atleast one conventional lipid comprises cholesterol, and whereincholesterol is present in an amount of between 10 and 45 mol % of thetotal lipid composition.

Aspect 53. The immunogenic composition of aspect 51, wherein thephosphatidylglycerol is present in an amount of between 20 and 80 mol %of the lipid composition.

Aspect 54. The immunogenic composition of aspect 51, wherein thephosphatidylserine is present in an amount of between 10 and 30 mol % ofthe lipid composition.

Aspect 55. The immunogenic composition of aspect 51, wherein the atleast one polar synthetic lipid comprises at least one syntheticimmunoactive glycolipid and at least one anionic lipid, and thearchaeosome further comprises at least one stabilizing lipid.

Aspect 56. The immunogenic composition of aspect 55, wherein the atleast one polar synthetic lipid comprises caldarchaeol having onecarbohydrate head group and one anionic head group.

Aspect 57. The immunogenic composition of aspect 56, wherein thecarbohydrate head group comprises gentiobiose and the anionic head groupcomprises phosphoinositol.

Aspect 58. The immunogenic composition of aspect 55, wherein the atleast one polar synthetic lipid comprises a first caldarchaeol havingtwo carbohydrate head groups and a second caldarchaeol having twoanionic head groups, and wherein the at least one stabilizing lipid isthe first and/or second caldarchaeol.

Aspect 59. The immunogenic composition of aspect 55, wherein the atleast one polar synthetic lipid comprises gentiotriose-archaeol andwherein the at least one stabilizing lipid comprises cholesterol and atleast one of phosphatidylethanolamine, archaetidylglycerol,archaetidylserine or archaetidylglycerolphosphate-methyl.

Aspect 60. The immunogenic composition of any one of aspects 41-59,wherein the composition comprises an HCV E1/E2 heterodimer.

Aspect 61. The immunogenic composition of aspect 60, wherein:

a) the HCV E2 polypeptide is derived from an HCV of major genotype 1, 2,3, 4, 5, 6, or 7; and

b) the HCV E1 polypeptide is derived from an HCV of major genotype 1, 2,3, 4, 5, 6, or 7.

Aspect 62. The immunogenic composition of any one of aspects 41-61,wherein the HCV E2 polypeptide comprises an amino acid sequence havingat least 20% amino acid sequence identity to an E2 polypeptide depictedin one of FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B.

Aspect 63. The immunogenic composition of any one of aspects 41-61,wherein the HCV E1 polypeptide comprises an amino acid sequence havingat least 20% amino acid sequence identity to an E1 polypeptide depictedin FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, and FIG. 4A-4B.

Aspect 64. The immunogenic composition of any one of aspects 41-63,wherein the E2 polypeptide and/or the E1 polypeptide lacks a C-terminaltransmembrane domain.

Aspect 65. The immunogenic composition of any one of aspects 41-64,wherein the HCV E2 polypeptide and the HCV E1 polypeptide are derivedfrom an HCV of the same genotype.

Aspect 66. The immunogenic composition of any one of aspects 41-64,wherein the HCV E2 polypeptide and the HCV E1 polypeptide are derivedfrom an HCV of different genotypes.

Aspect 67. The immunogenic composition of any one of aspects 41-66,wherein the HCV E1/E2 heterodimeric polypeptide comprises:

a) an HCV E1 polypeptide; and

b) a modified E2 polypeptide comprising, in order from N-terminus toC-terminus:

i) from 1 to 6 heterologous amino acids, wherein the from 1 to 6heterologous amino acids are C-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; and

ii) an HCV E2 polypeptide; or

a) an HCV E2 polypeptide; and

b) a modified E1 polypeptide comprising, in order from N-terminus toC-terminus:

i) from 1 to 6 heterologous amino acids, wherein the from 1 to 6heterologous amino acids are C-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; and

ii) an HCV E1 polypeptide; or

a) an HCV E1 polypeptide; and

b) a modified E2 polypeptide comprising, in order from N-terminus toC-terminus:

i) an HCV E2 polypeptide; and

ii) from 1 to 6 heterologous amino acids, wherein the from 1 to 6heterologous amino acids are N-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; or

a) an HCV E2 polypeptide; and

b) a modified E1 polypeptide comprising, in order from N-terminus toC-terminus:

-   -   i) an HCV E1 polypeptide; and    -   ii) from 1 to 6 heterologous amino acids, wherein the from 1 to        6 heterologous amino acids are N-terminal to a site of        proteolytic cleavage in a proteolytically cleavable linker.

Aspect 68. The immunogenic composition of aspect 67, wherein:

a) the from 1 to 6 heterologous amino acids at the N-terminus of themodified E2 polypeptide or the modified E1 polypeptide are Gly-Pro, Ser,Gly, or Gly-Ser; or

b) the from 1 to 6 heterologous amino acids at the C-terminus of themodified E2 polypeptide or the modified E1 polypeptide are LEVLFQGP (SEQID NO:117), ENLYYFQ (SEQ ID NO:150), LVPR (SEQ ID NO:124), I(E/D)GR (SEQID NO:125), or DDDDK.

Aspect 69. The immunogenic composition of any one of aspects 41-68,wherein the HCV E1 polypeptide, the HCV E2 polypeptide, or one or bothchains of the HCV E1/E2 heterodimer comprises a covalently linked T-cellepitope polypeptide comprising a T-cell epitope present in an HCVprotein other than E1 and E2.

Aspect 70. The immunogenic composition of any one of aspects 41-68,comprising a T-cell epitope polypeptide comprising a T-cell epitopepresent in an HCV protein other than E1 and E2, wherein the T-cellepitope polypeptide is not covalently linked to the HCV E1E2heterodimer, the HCV E1 polypeptide, or the HCV E2 polypeptide.

Aspect 71. The immunogenic composition of aspect 69 or aspect 70,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in one or more of:

a) an HCV non-structural polypeptide-3 (NS3) polypeptide;

b) an HCV non-structural polypeptide-2 (NS2) polypeptide;

c) an HCV non-structural polypeptide-4A (NS4A) polypeptide;

d) an HCV non-structural polypeptide-4B (NS4B) polypeptide;

e) an HCV non-structural polypeptide-5A (NS5A) polypeptide;

f) an HCV non-structural polypeptide-5B (NS5B) polypeptide;

g) an HCV core polypeptide; and

h) an HCV p7 polypeptide.

Aspect 72. The immunogenic composition of any one of aspects 69-71,wherein the T-cell epitope polypeptide has a length of from about 10amino acids to about 3000 amino acids.

Aspect 73. The immunogenic composition of any one of aspects 69-71,wherein the T-cell epitope polypeptide has a length of from about 10amino acids to about 50 amino acids, from about 100 amino acids to about230 amino acids, from about 230 amino acids to about 550 amino acids,from about 550 amino acids to about 780 amino acids, or from about 780amino acids to about 2000 amino acids.

Aspect 74. The immunogenic composition of any one of aspects 69-73,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in an HCV NS3 polypeptide.

Aspect 75. The immunogenic composition of aspect any one of aspects69-74, wherein the T-cell epitope polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to the aminoacid sequence of one of TP29, TP50, TP52, TP70, TP100, TP171, TP228,TP553, TP778, and TP1985.

Aspect 76. The immunogenic composition of aspect any one of aspects69-74, wherein the T-cell epitope polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to apolypeptide depicted in any one of FIGS. 9A, 9B, 10A-10D, and 11A-11N.

Aspect 77. The immunogenic composition of any one of 69-76, wherein theT-cell epitope polypeptide comprises one or more T cell epitopes presentin:

a) cholera toxin or toxoid; and/or

b) tetanus toxin or toxoid; and/or

c) diphtheria toxin or toxoid; and/or

d) CRM197.

Aspect 78. The immunogenic composition of any one of aspects 41-76,wherein the composition comprises a polypeptide comprising one or more Tcell epitopes present in:

a) cholera toxin or toxoid; and/or

b) tetanus toxin or toxoid; and/or

c) diphtheria toxin or toxoid; and/or

d) CRM197.

Aspect 79. A method of inducing an immune response to HCV in anindividual, the method comprising administering to the individual aneffective amount of the immunogenic composition of any one of aspects41-78.

Aspect 80. The method of aspect 79, wherein said administering is viaintramuscular administration, intranasal administration, subcutaneousadministration, or a combination thereof.

Aspect 81. The method of aspect 79, wherein said administering comprisesa prime and a boost.

Aspect 82. An immunogenic composition comprising:

a) one or more nucleic acids comprising nucleotide sequences encodingthe hepatitis C virus (HCV) E1 polypeptide, the HCV E2 polypeptide, orthe HCV E1/E2 heterodimer as recited in any one of aspects 41-78; and

b) an archaeosome as recited in any one of aspects 41-78.

Aspect 83. The immunogenic composition of aspect 82, wherein the one ormore nucleic acids are recombinant expression vectors.

Aspect 84. The immunogenic composition of aspect 83, wherein the one ormore recombinant expression vectors are recombinant viral vectors.

Aspect 85. The immunogenic composition of aspect 84, wherein the one ormore recombinant viral vectors are packaged into viral particles.

Aspect 86. The immunogenic composition of any one of aspects 82-84,wherein the one or more nucleic acids are present within non-pathogenicbacteria.

Aspect 87. A method of inducing an immune response to HCV in anindividual, the method comprising administering to the individual aneffective amount of any one of aspects 82-86.

Aspect 88. The method of aspect 87, wherein said administering is viaintramuscular administration, intranasal administration, subcutaneousadministration, or a combination thereof.

Aspect 89. The method of aspect 87, wherein said administering comprisesa prime and a boost.

Aspect 90. The method of any one of aspects 79-81 or aspects 87-89,wherein the immune response comprises one or more of a CD4⁺ response, aCD8⁺ response, and a neutralizing antibody response.

Aspect 91. The method of any one of aspects 79-81 or aspects 87-89,wherein the immune response induced is to more than one HCV genotype.

Aspect 92. An immunogenic composition comprising:

-   -   a) a T-cell epitope polypeptide comprising a T-cell epitope        present in a hepatitis C virus (HCV) protein other than E1 and        E2; and    -   b) a cyclic dinucleotide (CDN).

Aspect 93. The immunogenic composition of aspect 92, wherein the CDN isfluorinated.

Aspect 94. The immunogenic composition of aspect 93, wherein the CDN is2′-F-c-di-GMP.

Aspect 95. The immunogenic composition of aspect 92, wherein the CDN isof Formula

wherein:

A is S or O;

X is S, N, O, CH₂;

Y, Y′ is NH, CH₂, O;

Z, Z′ is NH, CH₂, O;

R1 represents hydrogen or NH₂ which may be substituted;

R2 is hydrogen or absent;

R3 represents NH₂, O, OH, H, or a halogen;

R4 represents hydrogen, halogen, or a straight or branched C₁-C₆ alkylgroup which may optionally be substituted;

R5 represents hydrogen, OH or a straight or branched C₁-C₆ alkyl chainor C₁-C₆ straight or branched alkoxy chain which may optionally besubstituted;

is a single or double bond;

or conjugates thereof, and salts or solvates thereof.

Aspect 96. The immunogenic composition of aspect 95, wherein the CDN isc-diGMP, c-diAMP, c-dilMP, or c-dXMP.

Aspect 97. The immunogenic composition of aspect 95, wherein the CDN isc-GpAp, c-Gplp, c-GpXp, c-Aplp, c-ApXp, or c-lpXp.

Aspect 98. The immunogenic composition of aspect 92, wherein the CDN iscyclic-GMP-AMP (cGAMP).

Aspect 99. The immunogenic composition of aspect 98, wherein the cGAMPis 2′3′-cGAMP, 2′2-cGAMP, 3′2′-cGAMP or 3′3′-GAMP.

Aspect 100. The immunogenic composition of any one of aspects 92-99,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in one or more of:

a) an HCV non-structural polypeptide-3 (NS3) polypeptide;

b) an HCV non-structural polypeptide-2 (NS2) polypeptide;

c) an HCV non-structural polypeptide-4A (NS4A) polypeptide;

d) an HCV non-structural polypeptide-4B (NS4B) polypeptide;

e) an HCV non-structural polypeptide-5A (NS5A) polypeptide;

f) an HCV non-structural polypeptide-5B (NS5B) polypeptide;

g) an HCV core polypeptide; and

h) an HCV p7 polypeptide.

Aspect 101. The immunogenic composition of any one of aspects 92-100,wherein the T-cell epitope polypeptide has a length of from about 10amino acids to about 3000 amino acids.

Aspect 102. The immunogenic composition of any one of aspects 92-100,wherein the T-cell epitope polypeptide has a length of from about 10amino acids to about 50 amino acids, from about 100 amino acids to about230 amino acids, from about 230 amino acids to about 550 amino acids,from about 550 amino acids to about 780 amino acids, or from about 780amino acids to about 2000 amino acids.

Aspect 103. The immunogenic composition of any one of aspects 92-102,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in an HCV NS3 polypeptide.

Aspect 104. The immunogenic composition of aspect any one of aspects92-103, wherein the T-cell epitope polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to the aminoacid sequence of one of TP29, TP50, TP52, TP70, TP100, TP171, TP228,TP553, TP778, and TP1985.

Aspect 105. The immunogenic composition of aspect any one of aspects1-20, wherein the T-cell epitope polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to apolypeptide depicted in any one of FIGS. 9A, 9B, 10A-10D, and 11A-11N.

Aspect 106. The immunogenic composition of any one of 92-105, whereinthe T-cell epitope polypeptide comprises one or more T cell epitopespresent in:

a) cholera toxin or toxoid; and/or

b) tetanus toxin or toxoid; and/or

c) diphtheria toxin or toxoid; and/or

d) CRM197.

Aspect 107. The immunogenic composition of any one of aspects 92-105,wherein the composition comprises a polypeptide comprising one or more Tcell epitopes present in:

a) cholera toxin or toxoid; and/or

b) tetanus toxin or toxoid; and/or

c) diphtheria toxin or toxoid; and/or

d) CRM197.

Aspect 108. A method of inducing an immune response to an antigen in anindividual, the method comprising administering to the individual aneffective amount of:

a) the composition of any one of aspects 92-107; and

b) the antigen.

Aspect 109. An immunogenic composition comprising:

a) a T-cell epitope polypeptide comprising a T-cell epitope present in ahepatitis C virus (HCV) protein other than E1 and E2; and

b) an archaeosome comprising at least one polar synthetic lipid, whereinthe at least one polar synthetic lipid comprises at least onecarbohydrate or anionic group linked by covalent bonding to at least onefree hydroxyl group of an archaeal core lipid.

Aspect 110. The immunogenic composition of aspect 109, wherein thearchaeal core lipid is archaeol (2,3-di-O-diphytanyl-sn-glycerol).

Aspect 111. The immunogenic composition of aspect 109, wherein thearchaeal core lipid is caldarchaeol(2,2′,3,3′-tetra-O-dibiphytanyl-sn-diglycerol).

Aspect 112. The immunogenic composition of aspect 109, wherein thecarbohydrate group is selected from the group consisting of:β-D-Glc-(1,6)-β-D-Glc-; β-D-Glc-(1,6)-.alpha.-D-Glc-;.alpha.-D-Glc-(1,6)-β-D-Glc-; β-D-Glc-(1,4)-β-D-Glc-;α-D-Glc-(1,4)-β-D-Glc-; β-D-Gal-(1,4)-β-D-Glc-; α-D-Gal-(1,6)-β-D-Glc-;β-D-Glc-(1,6)-β-D-Glc-(1,6)-β-D-Glc-;α-D-Glc-(1,4)-α-D-Glc-(1,4)-β-D-Glc-;α-D-Man-(1,2)-α-D-Man-(1,2)-α-D-Man-; andα-D-Man-(1,2)-α-D-Man-(1,2)-α-D-Man-(1,2)-α-D-Man-.

Aspect 113. The immunogenic composition of aspect 109, wherein thecarbohydrate group comprises two or three β-D-Glc-units in (1,6)linkage.

Aspect 114. The immunogenic composition of aspect 109, wherein thecarbohydrate group is a gal-glc-group.

Aspect 115. The immunogenic composition of aspect 109, wherein theanionic group is selected from the group consisting of phosphoserine,phosphoethanolamine, phosphoinositol and phosphoglycerol.

Aspect 116. The immunogenic composition of aspect 109, wherein the atleast one polar synthetic lipid comprises at least one anionic lipid.

Aspect 117. The immunogenic composition of aspect 116, wherein the atleast one anionic lipid is selected from the group consisting ofarchaetidylglycerol, archaetidylglycerolphosphate-methyl,archaetidylserine, and archaetidylinositol.

Aspect 118. The immunogenic composition of aspect 116, wherein thearchaeosome comprises at least one conventional lipid.

Aspect 119. The immunogenic composition of aspect 118, wherein the atleast one conventional lipid is selected from a group consisting ofphosphatidylglycerol, phosphatidylserine, SQDG, and cholesterol.

Aspect 120. The immunogenic composition of aspect 119, wherein the atleast one conventional lipid comprises cholesterol, and whereincholesterol is present in an amount of between 10 and 45 mol % of thetotal lipid composition.

Aspect 121. The immunogenic composition of aspect 119, whereinphosphatidylglycerol is present in an amount of between 20 and 80 mol %of the lipid composition.

Aspect 122. The immunogenic composition of aspect 119, whereinphosphatidylserine is present in an amount of between 10 and 30 mol % ofthe lipid composition.

Aspect 123. The immunogenic composition of aspect 109, wherein the atleast one polar synthetic lipid comprises at least one syntheticimmunoactive glycolipid and at least one anionic lipid, and thearchaeosome further comprises at least one stabilizing lipid.

Aspect 124. The immunogenic composition of aspect 123, wherein the atleast one polar synthetic lipid comprises caldarchaeol having onecarbohydrate head group and one anionic head group.

Aspect 125. The immunogenic composition of aspect 124, wherein thecarbohydrate head group comprises gentiobiose and the anionic head groupcomprises phosphoinositol.

Aspect 126. The immunogenic composition of aspect 123, wherein the atleast one polar synthetic lipid comprises a first caldarchaeol havingtwo carbohydrate head groups and a second caldarchaeol having twoanionic head groups, and wherein the at least one stabilizing lipid isthe first and/or second caldarchaeol.

Aspect 127. The immunogenic composition of aspect 123, wherein the atleast one polar synthetic lipid comprises gentiotriose-archaeol andwherein the at least one stabilizing lipid comprises cholesterol and atleast one of phosphatidylethanolamine, archaetidylglycerol,archaetidylserine or archaetidylglycerolphosphate-methyl.

Aspect 128. The immunogenic composition of any one of aspects 109-127,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in one or more of:

a) an HCV non-structural polypeptide-3 (NS3) polypeptide;

b) an HCV non-structural polypeptide-2 (NS2) polypeptide;

c) an HCV non-structural polypeptide-4A (NS4A) polypeptide;

d) an HCV non-structural polypeptide-4B (NS4B) polypeptide;

e) an HCV non-structural polypeptide-5A (NS5A) polypeptide;

f) an HCV non-structural polypeptide-5B (NS5B) polypeptide;

g) an HCV core polypeptide; and

h) an HCV p7 polypeptide.

Aspect 129. The immunogenic composition of any one of aspects 109-128,wherein the T-cell epitope polypeptide has a length of from about 10amino acids to about 3000 amino acids.

Aspect 130. The immunogenic composition of any one of aspects 109-128,wherein the T-cell epitope polypeptide has a length of from about 10amino acids to about 50 amino acids, from about 100 amino acids to about230 amino acids, from about 230 amino acids to about 550 amino acids,from about 550 amino acids to about 780 amino acids, or from about 780amino acids to about 2000 amino acids.

Aspect 131. The immunogenic composition of any one of aspects 109-130,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in an HCV NS3 polypeptide.

Aspect 132. The immunogenic composition of aspect any one of aspects109-131, wherein the T-cell epitope polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to the aminoacid sequence of one of TP29, TP50, TP52, TP70, TP100, TP171, TP228,TP553, TP778, and TP1985.

Aspect 133. The immunogenic composition of aspect any one of aspects109-132, wherein the T-cell epitope polypeptide comprises an amino acidsequence having at least 20% amino acid sequence identity to apolypeptide depicted in any one of FIGS. 9A, 9B, 10A-10D, and 11A-11N.

Aspect 134. The immunogenic composition of any one of 109-133, whereinthe T-cell epitope polypeptide comprises one or more T cell epitopespresent in:

a) cholera toxin or toxoid; and/or

b) tetanus toxin or toxoid; and/or

c) diphtheria toxin or toxoid; and/or

d) CRM197.

Aspect 135. The immunogenic composition of any one of aspects 109-133,wherein the composition comprises a polypeptide comprising one or more Tcell epitopes present in:

a) cholera toxin or toxoid; and/or

b) tetanus toxin or toxoid; and/or

c) diphtheria toxin or toxoid; and/or

d) CRM197.

Aspect 136. A method of inducing an immune response to an antigen in anindividual, the method comprising administering to the individual aneffective amount of:

a) the composition of any one of aspects 109-135; and

b) the antigen.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Induction of an Immune Response with HCV E1/E2 and a CDN oran Archaeosome

Various adjuvants including MF59, aluminum hydroxide/monophosphoryllipid A (Alum/MPLA), cyclic di-adenosine monophosphate (C-di-AMP) andarchaeosomes, were formulated with rE1E2 to immunize mice. 6-8 weeksmice were immunized three times on day 0, day 14, and day 35 and serawas collected two weeks after last immunization (FIG. 13). The abilityof the antibodies to neutralize HCV was evaluated using HCVpseudo-particle (HCVpp) carrying a luciferase reported gene, where theparticles were incubated with serum before culturing and the level ofluciferase expression was used to measure the neutralization activity ofthe given serum. Spleens were also collected, and isolated splenocyteswere restimulated ex vivo with short peptides to evaluate cellularimmunity using multicolor flow cytometry to detect intracellularproduction of cytokines. Different groups of mice received rE1E2formulated with MF59, Alum/MPLA, C-di-AMP, or archaeosomes along withappropriate controls

FIG. 13 depicts the immunization protocol.

Results

The Sera Collected from Immunized Mice is Able to Neutralize and Preventthe Entry of HCV Pseudo-Particles In Vitro:

As compared to the controls, the sera collected from immunized mice withadjuvanted rE1E2, showed significant increase in preventing the entry ofHCVpp in an in vitro neutralization assay (FIG. 14). When the antigenwas formulated with MF59 or Alum-MPLA, this effect was highly comparableto neutralization activity of anti-CD81 against HCVpp. However, thiseffect was lower than that of anti-CD81 in the case of C-di-AMP andarchaeosomes formulations. The route seemed to be playing a significantrole for C-di-AMP, since the sera from mice with three intranasaladministration (IN-IN-IN) of rE1E2+C-di-AMP did not neutralize HCVpp, asopposed to an intramuscular immunization followed by two intranasalboost (IM-IN-IN) regimen.

FIG. 14. Neutralization Activity of rE1E2 in Combination with Adjuvantin Mice.

The sera from mice immunized with rE1E2 were able to neutralize theentry of HCV pseudoparticle in vitro. Percentage of neutralization wascalculated based on the neutralizing activity for post-vaccination bleeddivided by neutralization activity of pre-vaccination bleed.

Horizontal lines are the medians in each group.

**=p value <0.01; *=p value <0.05.

Robust T Cell Immune Response in C-di-AMP and Archaeosomes Groups:

In vitro stimulation of mice splenocytes with a pool of 55 peptides thatspan the whole length of HCV E1E2 induced a strong memory T cellresponse in the groups, where the antigen was formulated with C-di-AMPand archaeosomes (p value <0.005). A moderate response was also detectedin Alum/MPLA group (p value <0.05). The data show that while C-di-AMPand archaesomes elicit strong neutralizing antibodies comparable to MF59and Alum/MPLA, both induced a more robust cellular immune response whichwas confirmed by the detection of vaccine-specific poly-functional CD4+T cells (FIG. 15).

FIG. 15. Differential Detection of T Cell Immune Response in Mice.

The splenocytes from vaccinated mice with rE1E2 in combination withdifferent adjuvants were stimulated in vitro and intracellularproduction of cytokine was detected by multi-color flow cytometry.IM=Intramuscular; IN=Intranasal; Neg=Negative control splenocytes;E1E2=Splenocytes that are stimulated with a pool of 55 peptides spanningE1E2.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. An immunogenic composition comprising: a) ahepatitis C virus (HCV) E1 polypeptide, an HCV E2 polypeptide, or an HCVE1/E2 heterodimer; and b) a cyclic dinucleotide (CDN).
 2. Theimmunogenic composition of claim 1, wherein the CDN is fluorinated. 3.The immunogenic composition of claim 2, wherein the CDN is2′-F-c-di-GMP.
 4. The immunogenic composition of claim 1, wherein theCDN is of Formula (I):

wherein: A is S or O; X is S, N, O, CH₂; Y, Y′ is NH, CH₂, O; Z, Z′ isNH, CH₂, O; R1 represents hydrogen or NH₂ which may be substituted; R2is hydrogen or absent; R3 represents NH₂, O, OH, H, or a halogen; R4represents hydrogen, halogen, or a straight or branched C₁-C₆ alkylgroup which may optionally be substituted; R5 represents hydrogen, OH ora straight or branched C₁-C₆ alkyl chain or C₁-C₆ straight or branchedalkoxy chain which may optionally be substituted;

is a single or double bond; or conjugates thereof, and salts or solvatesthereof.
 5. The immunogenic composition of claim 4, wherein the CDN isc-diGMP, c-diAMP, c-dilMP, c-dXMP, c-GpAp, c-Gplp, c-GpXp, c-Aplp,c-ApXp, or c-lpXp.
 6. The immunogenic composition of claim 1, whereinthe CDN is cyclic-GMP-AMP (cGAMP).
 7. The immunogenic composition ofclaim 6, wherein the cGAMP is 2′3′-cGAMP, 2′2-cGAMP, 3′2′-cGAMP or3′3′-GAMP.
 8. The immunogenic composition of claim 1, wherein the HCV E1polypeptide, the HCV E2 polypeptide, or one or both chains of the HCVE1/E2 heterodimer comprises a covalently linked T-cell epitopepolypeptide comprising a T-cell epitope present in an HCV protein otherthan E1 and E2.
 9. The immunogenic composition of claim 1, comprising aT-cell epitope polypeptide comprising a T-cell epitope present in an HCVprotein other than E1 and E2, wherein the T-cell epitope polypeptide isnot covalently linked to the HCV E1E2 heterodimer, the HCV E1polypeptide, or the HCV E2 polypeptide.
 10. The immunogenic compositionof claim 8, wherein the T-cell epitope polypeptide comprises an aminoacid sequence having at least 20% amino acid sequence identity to theamino acid sequence of one of TP29 (SEQ ID NO: 85), TP50 (SEQ ID NO:86), TP52 (SEQ ID NO: 87), TP70 (SEQ ID NO: 88), TP100 (SEQ ID NO: 89),TP171 (SEQ ID NO: 90), TP228 (SEQ ID NO: 91), TP553 (SEQ ID NO: 92),TP778 (SEQ ID NO: 93), and TP1985 (SEQ ID NO: 94).
 11. A method ofinducing an immune response to HCV in an individual, the methodcomprising administering to the individual an effective amount of theimmunogenic composition of claim
 1. 12. The method of claim 11, whereinsaid administering is via intramuscular administration, intranasaladministration, subcutaneous administration, or a combination thereof.13. The method of claim 11, wherein said administering comprises a primeand a boost.
 14. An immunogenic composition comprising: a) one or morenucleic acids comprising nucleotide sequences encoding a hepatitis Cvirus (HCV) E1 polypeptide, an HCV E2 polypeptide, or an HCV E1/E2heterodimer; and b) a cyclic dinucleotide.
 15. A method of inducing animmune response to HCV in an individual, the method comprisingadministering to the individual an effective amount of the compositionof claim
 14. 16. An immunogenic composition comprising: a) a hepatitis Cvirus (HCV) E1 polypeptide, an HCV E2 polypeptide, or an HCV E1/E2heterodimer, or one or more nucleic acids comprising nucleotidesequences encoding the HCV E1 polypeptide, the HCV E2 polypeptide, orthe HCV E1/E2 heterodimer; and b) an archaeosome comprising at least onepolar synthetic lipid, wherein the at least one polar synthetic lipidcomprises at least one carbohydrate or anionic group linked by covalentbonding to at least one free hydroxyl group of an archaeal core lipid.17. The immunogenic composition of claim 16, wherein the archaeal corelipid is archaeol (2,3-di-O-diphytanyl-sn-glycerol) or caldarchaeol(2,2′,3,3′-tetra-O-dibiphytanyl-sn-diglycerol).
 18. A method of inducingan immune response to HCV in an individual, the method comprisingadministering to the individual an effective amount of the immunogeniccomposition of claim 16.